Journal of Toxicology and Environmental Health, Part A

Current Issues

ISSN: 1528-7394 (Print) 1087-2620 (Online) Journal homepage: https://www.tandfonline.com/loi/uteh20

Acute and chronic toxicity assessment of

haloacetic acids using Daphnia magna

Armindo Melo, Cláudia Ferreira, Isabel M.P.L.V.O. Ferreira & Catarina

Mansilha

To cite this article: Armindo Melo, Cláudia Ferreira, Isabel M.P.L.V.O. Ferreira & Catarina
Mansilha (2019): Acute and chronic toxicity assessment of haloacetic acids using Daphnia�magna,
Journal of Toxicology and Environmental Health, Part A, DOI: 10.1080/15287394.2019.1676959

To link to this article: https://doi.org/10.1080/15287394.2019.1676959

Published online: 12 Oct 2019.

Submit your article to this journal

Article views: 3

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=uteh20
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A
https://doi.org/10.1080/15287394.2019.1676959

Acute and chronic toxicity assessment of haloacetic acids using Daphnia magna
Armindo Meloa,b, Cláudia Ferreiraa, Isabel M.P.L.V.O. Ferreirac, and Catarina Mansilhaa,b
a
Departamento de Saúde Ambiental, Instituto Nacional de Saúde Doutor Ricardo Jorge, 4000-055 Porto, Portugal; bLAQV/REQUIMTE - Universidade
do Porto, Porto, Portugal; cLAQV/REQUIMTE/Departamento de Ciências Químicas, Laboratório de Bromatologia e Hidrologia, Faculdade de Farmácia -
Universidade do Porto, Porto, Portugal

ABSTRACT KEYWORDS
Haloacetic acids (HAAs) are undesirable disinfection by-products (DBPs), released into aquatic Daphnia magna; disinfection
ecosystems from various anthropogenic and natural sources. The aim of this study was to examine by-products; wastewater;
the ecological risk of exposure to three HAAs commonly detected in water, such as monobro- haloacetic acids; ecotoxicity
moacetic acid (MBA), monochloroacetic acid (MCA), and trichloroacetic acid (TCA), in in vivo acute
and chronic toxicity tests using Daphnia magna as a model. Acute tests showed that MBA was the
most toxic of these compounds followed by MCA and TCA as evidenced by immobilization.
Aquatic organisms in natural conditions might be exposed simultaneously to numerous com-
pounds; thus, binary mixtures of selected HAAs and a ternary mixture of these were tested.
Concentration addition (CA) and independent action (IA) models were used for a predictive
assessment of mixture toxicity. Data demonstrated that CA appeared to be the most reliable
indicator for HAAs binary and ternary mixtures suggestive of an additive behavior. Median
effective concentration (EC50) values from the mixed exposure tests were significantly lower
than results obtained from single tests for all three HAAs where an increase of toxicity greater
than 50%. Multigenerational chronic tests were also performed exposing daphnids to the ternary
mixture of HAAs. A markedly decreased sexual maturity and number of offspring and broods per
daphnid especially in the second generation were noted.

Introduction reactions between natural organic matter (NOM)

and chlorine, or other disinfectants, such as chlora-
Water and wastewater disinfection procedures are
mines or ozone, mainly during water treatment pro-
recognized as one of the most important and ben-
cedures (Boorman et al. 1999; Hong, Song, and
eficial public health advances of the 20th century,
Karanfil 2013; Kim et al. 2003; Krasner et al. 2009;
due to their contribution to reduce the incidence
Sirivedhin and Gray 2005). HAAs are also produced
of diseases produced by waterborne pathogens
during industrial processes, such as textile finishing
(CDC 1999). Despite being used for over 100
and herbicidal formulations (Berg et al. 2000), or
years, there has been a concern that while disin-
result from natural sources as algae activities
fection provides a precious protection against
(Huang et al. 2008). Several investigators reported
microbial risks, this procedure may also pose che-
that the discharge of effluents from wastewater treat-
mical risks associated with the formation of harm-
ment plants (WWTPs) might be the major source of
ful contaminants, namely disinfection by-products
HAAs in nature (Krasner et al. 2009; Muller et al.
(DBPs) (Simmons et al. 2008; WHO 2016).
1996; Rebhun, HellerGrossman, and Manka 1997).
Haloacetic acids (HAAs) constitute one of the
Therefore, HAAs are ubiquitous not only in tap
major classes of DBPs, together with trihalo-
water systems and swimming pools, but also in
methanes (Krasner et al. 2006; Richardson et al.
aquatic ecosystems (Berg et al. 2000; Muller et al.
2008). Regarding their chemical composition,
1996; Scott et al. 2005).
HAAs are a group of several different halogenated
The concentration of trichloroacetic acid (TCAA)
carboxylic acids, containing nine compounds that
was measured in various water sources as summar-
include chlorine and/or bromine in their structure
ized by IARC (IARC 2004). Concentrations varied
(Richardson et al. 2008). These arise from chemical

CONTACT Catarina Mansilha [email protected] Departamento de Saúde Ambiental, Instituto Nacional de Saúde Doutor Ricardo
Jorge, Rua Alexandre Herculano, 321, Porto, 4000-055, Portugal.
© 2019 Taylor & Francis Group, LLC
2 A. MELO ET AL.

from 0.05 µg/L in groundwater distribution systems et al. 1996; Scott et al. 2002). Therefore, in order to
in Germany, to 200 µg/L in chlorinated water in prevent the underestimation of its real impact in
Australia (WHO 2004). Despite the lower concen- ecosystems, toxicity assays also need to be conducted
tration of monobromoacetic acid (MBAA) and with mixtures rather than using only single contami-
monochloroacetic acid (MCAA) in natural and trea- nants (Bopp et al. 2019; Heys et al. 2016; Kar and
ted waters, data demonstrated that these chemicals Leszczynski 2019; Kienzler et al. 2016).
are more toxic than their di- and tri-analogs. This is Computational approaches are not a substitution
of particular interest with respect to the toxicity for in vivo and in vitro toxicity tests, but might
impact on aquatic species even after short-term complement experiments by minimizing animal
exposure (Fisher et al. 2014; Giller et al. 1997). testing, decreasing cost and time of toxicity tests, as
Several regulatory limits exist for drinking water well as improving toxicity prediction and risk assess-
established by organizations such as the US ment (Kar and Leszczynski 2019). The concentration
Environmental Protection Agency (US EPA) (EPA addition (CA) (Loewe and Muischnek 1926) and
2009) and World Health Organization (WHO) independent action (IA) (Bliss 1939) are two differ-
(WHO 2017), which defined maximum contami- ent theories that describe mixture effects for similar-
nant levels for some HAAs based upon potential and dissimilar-acting toxicants, respectively. One
carcinogenic effects observed due to long-term expo- common trait of the two models is that these assume
sure. In Europe, despite no guideline values set for non-interaction as the default. Deviation from the
HAAs in the Drinking Water Directive 98/83/EC prediction is thus an indication of interaction (antag-
(DWD), countries like the United Kingdom recom- onistic (weaker) or synergistic (stronger) effects than
mended that these compounds should be included as predicted) (Cedergreen et al. 2008; Syberg et al.
a ‘high priority’ in the list of regulatory chemical 2008).
parameters to be routinely monitored (Malliarou In most cases, the concentration of a chemical
et al. 2005). Further, some HAAs were also classified in the environment is far below its individual
by the International Agency for Research on Cancer median effective concentration 50% (EC50), or
(IARC) as possibly carcinogenic to humans (Group even below its individual no observed effect con-
2B), and with sufficient evidence in experimental centration (NOEC). However, even at low concen-
animals for carcinogenicity (IARC 2013, 2014) trations, a compound might still significantly alter
Animal studies demonstrated that exposure to the adverse effect of the mixture of which it forms
HAAs might produce adverse effects on liver, kid- a part. In order to estimate a more realistic thresh-
ney, nervous system, male reproductive effects, and old of response, lab-tested mixtures need to be
fetal malformations (Acharya et al. 1995; EPA 1988; present at low chemical concentrations, similar to
Herrenfreund et al. 1987; Narotsky et al. 2011). It those observed in aquatic systems, but able to
should be noted that (Narotsky et al. 2008) reported initiate a response. One should not perform only
that exposure to water disinfected by chlorination one point experiment for the mixture, but measure
and ozonation/postchlorination did not markedly the dilution series of the mixture to enable
affect the developmental outcome in Sprague– a design of a complete dose-response curve.
Dawley rats. Despite the global information regard- Further, given the continuous entry and formation
ing HAAs toxicity, little is known with respect to of HAAs in aquatic environments, chronic studies
potential adverse impact on aquatic animals that are are also important to examine the long-term
may be exposed to DBPs a greater extent than ter- effects of pollutants on test model organisms
restrial ones, as chemical uptake is not only through including full life-cycle investigations, which may
the gastrointestinal pathway, but continuously detect sublethal and late-life effects, which may be
through large body surfaces such as skin and, in missed in short-term tests. Similarly, most eco-
particular, gills (WHO 2016). toxicological test guidelines assess the influence
In natural environment, HAAs may coexist, observed within one generation, in which the
resulting in multiple interactions that might alter potential detrimental effect that may require dif-
their exposure-related outcomes (Berg et al. 2000; ferent generations to occur is overlooked (Barata
Hanson and Solomon 2004; Kim et al. 2003; Muller et al. 2017), as for certain compounds/mixtures
 JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 3

severity effects are exhibited only in subsequent was renewed 20% three times a week. Daphnids were
generations (Campos et al. 2016). fed daily with a concentrated suspension of the green
Among the existing ecotoxicological assays, the algae Chlorella vulgaris (standard value of 3 × 105
Daphnia magna test is probably the most cells/ml/daphnia) and an organic additive
employed and standardized test in aquatic toxicol- (Marinure, seaweed extract, supplied by Glenside
ogy. This crustacean is approved as model for Organics Ltd.) at a ratio of 4 ml per 800 ml ASTM
toxicity tests by several international organizations medium (Baird et al. 1989). The green alga was resus-
dealing with toxicity testing procedures in view of pended in the ASTM medium in order to avoid the
its application within a regulatory framework transfer of algae growth medium to culture vessels of
(EPA 2002; OECD 1984, 2004, 2012; Sancho, daphnia (ASTM 2004; ISO 2012).
Villarroel, and Ferrando 2016) due to its high To probe the reliability of animal responses to
sensitivity to pollutants, short life span, rapid toxicity tests, reference studies with potassium
reproduction, and maintenance in lab. dichromate were conducted in accordance to ISO
The aim of this study was to examine the 6341 (ISO 2012) guideline (ISO 2012).
adverse effects of three HAAs (TCAA, MCAA, International inter-lab trials of LGC Standards
and MBAA), known to be harmful to aquatic (Sample 50 of Aquacheck Scheme) were also per-
organisms, as individual chemicals as well as mix- formed for EC50 24 and 48hr determinations.
tures, in both acute and chronic toxicity tests using
Daphnia magna.
Chemicals and reagents
Monochloroacetic acid (MCA), trichloroacetic acid
Materials and methods (TCA), and monobromoacetic acid (MBAA) were
purchased from Sigma-Aldrich (Steinheim,
Test species
Germany). Sodium nitrate and potassium dichro-
D. magna individuals (clone A, sensus) (Baird et al. mate (K2Cr2O7) used as reference substance and
1989) were obtained from the 3rd to 5th brood neo- ethanol ≥99% were procured from Merck
nates from a continuous culture maintained in the lab (Darmstadt, Germany). Ultra-pure water (0.054 μS/
following the recommendations of standard protocols cm) was obtained by using a Milli-Q gradient system
(EANLS 2007; ISO 2012). ASTM synthetic hard water from Millipore (Milford, MA, USA). Chemical for-
(ASTM 2004) was used as culture medium for mulas, molecular weights, purities, and concentra-
D. magna (nominal components: NaHCO3 = tion ranges used are presented in Table 1. Individual
0.19 mg/L; MgSO4.7H2O = 245 mg/L; KCl = 8 mg/L; trace elements and macronutrients of ASTM culture
CaSO4 · 2H2O = 120 mg/L; pH 8.2–8.4; hardness medium were purchased from Merck. Individual
160–180 mgCaCO3/L; and alkalinity 110–120 stock standard solutions of the studied compounds
mgCaCO3/L). Animals were cultured at were prepared in ASTM medium by weighing of
a temperature of 20°C and a 16:8 hr light:dark photo- high purity standards and accurate dilution.
period. The rearing procedures followed the (OECD 2012).
Organization for Economic Co-operation and The concentrated suspension of green algae
Development (OECD) recommendations of standard Chlorella vulgaris was grown in Woods Hole MBL
protocols (OECD 2004, 2012). Stock population den- medium supplied with all necessary macro- and
sity was 20 individuals per 1000 ml medium, which micronutrients (Nichols 1979). The macronutrients

Table 1. Chemical compounds used in the single-compound acute toxicity tests.

Concentrations
Chemical Cas No. Molecular weight Purity (Min-Max)
Potassium Dichromate (K2Cr2O7) 7778-50-9 294.19 g/mol 99.9 % 0.2–3.2 mg/L
Bromoacetic acid (C2H2BrO2) 79-08-3 138.948 g/mol 97% 10 – 50 mg/L
Chloroacetic acid (C2H3ClO2) 79-11-8 94.50 g/mol 99% 80 – 200 mg/L
Trichloroacetic acid (C2HCl3O2) 76-03-9 163.387g/mol ≥99% 200 – 400 mg/L
4 A. MELO ET AL.

employed on MBL culture medium were calcium multi-component mixtures. The toxicity parameter
chloride dihydrate (CaCl2.2H20), magnesium sulfate used for Daphnia magna was 48hr immobilization,
heptahydrate (MgSO4.7H2O), sodium bicarbonate termed median effective concentration (EC50). The
(NaHCO3), dipotassium phosphate (K2HPO4), concentrations employed in the single-compound
sodium nitrate (NaNO3), and sodium metasilicate acute toxicity tests were defined in accordance with
pentahydrate (NaSiO3.9H2O) from Merck preliminary testing results also performed in our lab
(Darmstadt, Germany) and from Sigma-Aldrich under the same conditions as described above.
(Steinheim, Germany). The micronutrients ferric A geometric series of 6–8 concentrations was identi-
chloride hexahydrate (FeCl3.6H2O), copper (II) sul- fied for each compound. The minimum and max-
fate pentahydrate (CuSO4.5H2O), zinc sulfate imum concentrations tested are presented in Table 1.
heptahydrate (ZnSO4.7H2O), cobalt chloride hexahy- Concerning the acute toxicity analyses of differ-
drate (CoCl2.6H2O), manganese chloride tetrahydrate ent HAAs mixtures, 6–8 concentrations were also
(MnCl2.4H2O), sodium molybdate dehydrate tested following a geometric series of dilutions
(NaMoO3.2H2O), disodium ethylenediaminetetraace- (factor 1/8). In this case, the total concentration
tate dihydrate (Na2-EDTA), and tris(hydroxymethyl) varied while the concentration ratio of the HAAs
aminomethane were also purchased from Merck was constant (so-called fixed ratio design) (Greco,
(Darmstadt, Germany) and from Prolabo (Belgium). Bravo, and Parsons 1995; Sorensen et al. 2007).
The vitamins thiamine, cyanocobalamine, and biotine The compounds were mixed in ratios correspond-
were procured from Sigma-Aldrich (Steinheim, ing to 50:50% for binary mixtures and a 33:33:33%
Germany). effect ratio for ternary mixtures. Experiments were
designed to enable a valid statistical estimation of
concentration-response functions in the range
Experimental design and test method
between EC1 and EC99. Initially, three binary
The particular and cumulative effects of selected HAAS mixtures that combine MBAA+MCAA,
substances were assessed according to OECD MBAA+TCAA, and MCAA+TCAA, and
guidelines. Acute toxicity experiments followed a mixture of them all (Mix3HAAs) were utilized.
the OECD 202 protocol for testing chemicals in Chronic toxicity tests were conducted according
D. magna (OECD 2004). Neonates, aged less than to the OECD 211 guidelines (OECD 2012) to
24hr, were placed in glass test vessels containing assess the chronic ecotoxicity of the ternary mix-
25 ml of different concentrations of test solutions ture. Less than 24hr neonates from a second brood
for a period of 48hr. Four replicates (5 neonates progeny were placed individually in glass vessels
per test chamber) per test concentration level and (100 ml of test solution) and incubated with test
untreated control medium were prepared. Vessels substances at a temperature of 20ºC and a 16:8 hr
were maintained at a temperature of 20ºC and light:dark photoperiod for 21 days. Ten replicates
a 16:8 hr light:dark photoperiod. No feeding was per test and untreated control medium were pre-
provided during the test. Tests were considered pared. Concentrations of 10% of the EC50 values of
acceptable only if control survival was ≥90%. each HAA calculated from Mix3HAAs (Table 2)
Concentration-response analyses were performed maintaining the proportion of 33:33:33% were
in essentially the same manner for all single and selected, as previous studies performed with higher

Table 2. Estimated EC50 values for D. magna 48 h acute toxicity tests and inferior and upper limits of the 95% confidence interval
(CI) from dose–response curves fitted via Probit test.
EC50 (48h, 95%CI) mg/L; [Inferior limit – Upper limit]
Solution MBAA MCAA TCAA
Single compound solution 18.8 [16.6–20.8] 74.2 [42.4–99.2] 353.9 [347.1–366.3]
Mix MBAA + MCAA 6.7 [3.4–9.4] 33.4 [17.2–47.0] –
Mix MBAA + TCAA 13.2 [12.3–14.0] – 239.6 [226.0 − 253.0]
Mix MCAA + TCAA – 47.8 [44.4–51.2] 172.0 [159.9–184.1]
Mix3HAAs 7.9 [7.1–8.6] 39.3 [35.4–42.7] 141.4 [127.3–153.9]
 JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 5

concentrations resulted in a mortality of animals X

n
ci
that exceeded the 50% at the start of the tests. ¼1
i¼1
Ei
In each vessel, the renewal of the test solution
was performed 3 times per week. Daphnids were In the above equation, ci are the concentrations of
fed daily with concentrated green algae Chlorella the individual substances present in a mixture with
vulgaris suspensions with ration levels of between a total effect of x%. Ei are the equivalent effect
0.1 and 0.2 mg C/Daphnia/day. Each day, data concentrations of the single substances, i.e. those
from the parent survival, molting frequency, and concentrations that alone would produce the same
number of newborns were collected. Newborns quantitative effect x as the mixture. If the left-hand
were removed from the vessels. Tests were consid- side of the equation is less than one, there is
ered valid if in control vessels the mortality of the a synergistic effect, and if the left-hand side is
parents did not exceed 20%, and surviving off- greater than one, the effect is antagonistic (Faust
spring produced per daphnia were higher than 60 et al. 2001).
neonates at the end of the experiment. The alternative concept of IA is based upon the
A second-generational test was also performed. idea of a dissimilar action of mixture components
Normally developed neonates (maximum age 24hr and is given by the equation:
at the start of the test), originated from the third
clutch of offspring of daphnids from the reproduction Y
n
Eðcmix Þ ¼ 1  ð1  Eðci ÞÞ
test, hereafter referred to as parental generation or i¼1
first-generation. These daphnids were treated with
selected test concentrations during embryonic devel- in which ci and cmix are the concentrations of the
opment and further exposed to the same concentra- individual constituents and the total concentration
tions of test substances. Individuals from of the mixture, respectively. E(ci) denotes the corre-
the second-generation were maintained similar to sponding effects of the individual constituents and
the parental one for 21 days, following also the E(cmix) the total effect of the mixture. Effects E are
OECD guidelines (OECD 2012). Neonates from expressed as fractions (x%) of a maximum possible
unexposed control organisms were maintained in an effect (Faust et al. 2001).
uncontaminated medium. Experiments were con- To assess potential deviations of the experimen-
ducted on 10 replicates (Barata et al. 2017). Several tal toxicities from predictions, the accuracy of the
physical and chemical parameters of ASTM medium predictive approaches was quantified by applying
were analyzed including dissolved oxygen concentra- the Model Deviation Ratio (MDR) (Belden,
tion, conductivity, pH, and temperature at the begin- Gilliom, and Lydy 2007), to the CA and IA mod-
ning, each time the medium was renewed and at the els. MDR is defined as:
end of acute and chronic tests. HAAs were quantified Expected Predicted ECx mix
in a Shimadzu GCMS-QP2010 Gas Chromatograph MDR ¼ ¼
Observed Observed ECx mix
Mass Spectrometer equipment during tests.
where Expected is the effective concentration of
the mixture that would be predicted by the
Prediction of mixture toxicities
model and Observed is the experimentally
Once the responses of individual chemicals were observed effect concentration.
established, the toxic effects of the mixtures were A MDR of 1 characterizes perfect compliance
subsequently predicted using the concentration between predicted and observed toxicity of the
addition (CA) and independent action (IA) mod- mixture. A MDR of greater than 1 indicates that
els. The experimentally derived effects were then the model underestimates toxicity, while a value
compared with the predicted ones. of less than 1 indicates that the model overesti-
The concept of CA is based upon the assump- mates toxicity. MDR values ranging from 0.5 to 2
tion that the components of a given mixture act (0.5 ≤ MDR ≤ 2) indicate a compliance with the
similarly and the effective concentration can be predictive approaches (Belden, Gilliom, and Lydy
predicted from the equation: 2007). MDR values were calculated for all effect
6 A. MELO ET AL.

levels (ECx) for which the respective estimates for enabled confirming their persistence over various
the single substances had been determined. time intervals with recovery results between 80%
and 120%. Further, the EC50,24hr value for the
reference acute test performed with potassium
Statistical analyses dichromate was 0.8 mg/L, which is within the
Data of the physical–chemical parameters in the estimated range of 0.6–2.1 mg/L (ISO 2012).
ASTM medium between replicate chambers in Participation in international inter-lab trials of
controls and test vessels were subjected to one- LGC Standards (Sample 50 of Aquacheck
way analysis of variance (ANOVA). Scheme) was also performed with satisfactory
The EC50 values were calculated using data from results (z-scores of 0.9 ± 0.37 for Daphnia magna
the 48hr acute toxicity tests (concentration esti- EC50,48hr and z-scores of 1.25 ± 0.59 for Daphnia
mated to immobilize 50% of the daphnids within magna EC50,24hr, n = 4), affirming that the stipu-
a stated exposure period). Daphnids unable to lated lab conditions were successfully applied.
swim for 15sec after stirring were considered
immobile, and % of immobilization relating to
either control medium or solvent control were Acute immobilization tests
used to estimate EC50 with log-logistic concentra- The EC50 values obtained from MBAA, MCAA, and
tion effect curves. The dose–response curves were TCAA single-chemical exposure tests are presented
fitted via the Probit analysis. in Table 2, along with data attained in the mixture
EC50 values and statistical tests were performed experiments. Control mortality was zero and con-
using SPSS version 25.0 Statistics Inc. for windows, trols did not show any behavioral abnormalities.
Chicago, IL, USA. CA and IA predictions were A considerable variability of toxicity was found
carried out using the mixtox package in among HAAs with MBAA being the most toxic to
R (https://cran.r-project.org/web/packages/mix D. magna with an EC50 value of 18.8 mg/L, approxi-
tox/index.html). mately 4- and 19-fold more potent than EC50 of
Regarding predictive model tests, the expected MCAA and of TCAA, respectively.
and observed EC50 values were compared for sig- Mixture solutions were prepared as described
nificance using Dunnett’s test. Statistical signifi- above. Data demonstrated that the EC50 values of
cance was set at p < .05. each HAA in the tested mixtures were significantly
lower than levels observed for the isolated com-
pounds (MBAA, MCAA, and TCAA as single solu-
Results
tions). The enhanced toxicity of approximately 55%
The maintenance of physico-chemical properties of suggested that toxicologically significant interactions
the exposure media was ensured during acute and occur at different concentration levels (Table 2).
chronic tests, both in control and test vessels indicat- The overall toxicity of the mixtures was also cal-
ing that daphnids' responses were not markedly culated and data presented in Table 3. Exposure to
affected by medium composition variance. All fresh Mix3HAAs resulted in lower quantities required to
and old media presented constant culturing condi- produce immobilization compared to the binary
tions, with values of temperature of 20 ± 2ºC, pH 7.29, mixtures with the exception of MBAA+MCAA.
electrical conductivity 535 ± 85 µS/cm, and dissolved The presence of the monohaloacetic forms appeared
oxygen 6.46 ± 0.21 mg/L, which is in accordance with to confer a significant rise in the toxicity of the
the validation criteria of OECD 202 (OECD 2004) mixture. The predictability of mixtures toxicities
and OECD 211 (OECD 2012) guidelines. based upon findings from single-chemical para-
HAAs concentrations were also measured at the meters was evaluated on data obtained from CA
beginning and immediately prior to culture med- and IA over their entire effect ranges. Results are
ium renewal or test termination. The values in old provided in Table 3, where observed experimental
media were similar to the initial ones, which data and estimated CA and IA values are presented.
 JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 7

Table 3. Summary of EC50 predicted and observed effect con- The concentration–response curves (CRCs) illus-
centrations for mixtures under study. trate that CA describes the data reliably over the
EC50 (48h, 95%CI) mg/L; [Inferior limit – entire effect range (Figure 1 – A1, A2, A3, A4).
Upper limit]
Solution Mixture CA IA
CRCs of mixtures demonstrate good agreement
Single compound solution – – – with predictions without deviation from the concep-
Mix MBAA + MCAA 40.1 [20.6–56.4]* 59.2 107.2 tual expectation. Similar conclusions were obtained
Mix MBAA + TCAA 252.9 [238.5–267.0]* 244.9 456.4
Mix MCAA + TCAA 228.8 [224.3–233.4]* 225.4 439.6
through the analyses of MDR values that assessed the
Mix3HAAs 183.7 [171.3–197.0] 184.9 356.4 compliance (or deviation) of the observed toxicities
* Significant from Mix3HAAs (p < 0.05). with CA and IA predictions (Figure 2).

Figure 1. Observed and predicted Daphnia magna toxicity of mixtures under study. (A1) MBAA + MCAA; (A2) MBAA + TCAA; (A3)
MCAA + TCAA; (A4) Mix3HAAs. (•) Experimentally observed toxicity; (dashed line –) prediction according to concentration addition;
(solid line –) prediction according to independent action.

Figure 2. Model Deviation Ratio (MDR) for CA (A) and IA (B) for Daphnia magna toxicity (ECx) of mixtures under study.
8 A. MELO ET AL.

For HAAs binary and ternary mixtures, which the test group, in any generation. The mean num-
were best modeled by CA, MDR values lie between ber of live offspring produced per control parent
0.77 and 1.46, indicating an additive behavior of animal was greater than 60, which enabled valida-
the single compounds at all effect levels (0.5≤ tion of the test according to the TG 211 biological
MDR≤2). Lower compliance with the IA assump- criteria (OECD 2012). A control test was also
tions was obtained with MDR values varying from performed for the 2nd generation, with similar
1.77 to 2.62 at a 50% effect level. In this case, results.
deviation from prediction was noted and toxicity Concerning the first generation, the cumulative
was underestimated (MDR values near or number of neonates per female underwent no sig-
above 2). nificant differences between test and control with
offspring mean values of 104 and 118, respectively
(Figure 3). However, in the following generation,
Multigenerational tests the number of offspring per female was signifi-
A 21-day chronic trial was performed according to cantly reduced with a mean value of 28 neonates
OECD Test Guideline 211 (TG211) to determine in test and 106 in control.
the biological impact of Mix3HAAs on D. magna Under ideal physicochemical conditions,
(OECD 2012). Recognizing that toxicant pulses D. magna reproduces parthenogenetically, produ-
may affect daphnids differently during their life cing clutches of eggs every 3 to 4 days until its
cycle, a second-generational chronic toxicity test death. The age at which the first eggs are deposited
was also performed. No adult mortality occurred into the brood chamber is around 5–10 days at
in the control or mixture solution vessels over the 20ºC and then released as free-swimming neo-
21-day testing period. In addition, no male off- nates. It was found that in the second-generation
spring was produced in the control group, or in test, a later sexual maturity occurred with the first

Figure 3. Multigenerational chronic test. Mean cumulative number of neonates produced by two generations of Daphnia magna
exposed to a mixture of 10% of the EC50 values calculated for MBAA, MCAA, TCAA and control (n = 10).
 JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 9

Figure 4. Multigenerational chronic test. Effects of a mixture of 10% of the EC50 values of MBAA, MCAA and TCAA on brood sizes
over two generations of D. Magna (n = 10). * Significant difference (p < .05).

eggs released only after day 13. The number of broods was reported by Kühn et al (1989a) for a 24hr
per daphnia was also significantly reduced from 6 in exposure period, suggesting that for 48hr lower
control to 5 in the first generation, to 4 broods after concentrations may exert a negative impact.
continuous exposure to HAAs (Figure 4). Results also Regarding MCAA the EC50,48hr value of 74.2 mg/
show that in the 1st generational chronic toxicity test L was also similar to levels reported by Kühn et al
and control, the highest average number of offspring (1989b). Kuhn et al. (1989b) found an EC50 for
per daphnia was reached in the 4th brood, corre- 24hr was 99 mg/L and an EC50 for 48hr was
sponding to 34 and 40 neonates, respectively. 77 mg/L. TCAA tests required an elevation of the
However, in the 2nd generation, this value reached range of concentrations in order to attain an effect
only 8 neonates in the 4th brood the highest mean in daphnids indicating lower toxicity (EC50 value
value of 11 neonates obtained in the 1st brood. of 353.9 mg/L). This is also in agreement with the
lower genotoxicity and less toxicity in freshwater
algae reported in previous studies (Richardson
Discussion
et al. 2007; Roberts, van Egmond, and Price 2010).
The EC50 values of MBAA, MCAA, and TCAA The prediction of combined effects based upon
single solutions demonstrated different acute curve fitting information of individual compounds
effects with MBAA the most toxic compound, might be a promising tool for the risk assessment
which is in agreement with the evidence of of pollutants mixtures, which is of great impor-
a major toxicity of the brominated HAAs over tance for regulatory agencies. For HAAs mixtures,
their chlorinated analogs (Plewa et al. 2002). Inter- the CA reference model provided the better case
lab trials and comparison of our acute toxicity data estimation for the joint effect, just as expected
with literature reinforce the confidence in the nor- from the theory of “same mode of action”.
mal behavior and sensitivity of D. magna to che- In the chronic toxicity test, the concentrations of
micals. Overall, the 48 hr EC50 values obtained in HAAs to which our test organism was exposed were
the present study were in accordance with similar lower than those used in acute tests, although above
studies. For MBAA, a value of 65 mg/L of EC50 the levels reported in water quality monitoring
10 A. MELO ET AL.

studies (Berg et al. 2000; IARC 2004; Muller et al. Since anthropogenic activity is continuously
1996; Scott et al. 2005). Nevertheless, the current altering aquatic systems through direct or indirect
trend is for DBPs concentrations to increase in contaminations, the possibility of bioaccumulation
natural and treated waters and, consequently, envir- can not be ruled out. Chronic studies using more
onmental burdens may result in potential long-term environmentally realistic concentrations of pollu-
ecological risks to aquatic biota. tants showed that the effects of HAAs were more
Adverse reproductive effects were noted espe- noticeable in the second generation of D. magna
cially in the second generation. A diminished ability with a disruption of their reproductive process.
of the descendants to tolerate these compounds was Hence, it is anticipated that aquatic organisms
detected reinforcing the idea that the extrapolation may not always evolve a resistance to pollutants
from acute to chronic effects always needs to be in natural systems, presumably due to high fitness
done with caution. Thus, in current regulatory prac- costs. Therefore, one thus cannot exclude the
tice, the No Observed Adverse Effect Levels harmful effects attributed to these DBPs in aquatic
(NOAEL) are combined with uncertainty factors, ecosystems. Further research is needed to extend
as species–species extrapolations, inter-individual our knowledge regarding the influence of these
variations and extrapolations from acute to chronic pollutants in the environment, especially of com-
effects (Kortenkamp, Backhaus, and Faust 2009). plex mixtures on non-target organisms in order to
It is difficult to predict the real impact of develop a more accurate risk assessment.
a mixture of HAAs, even at low concentrations,
in aquatic ecosystems. However, our results sug-
gest that these compounds might effectively affect Acknowledgments
small invertebrates like daphnia, and thus, may The authors are greatly indebted to all financing sources
compromise the nutritional composition of the (FEDER POCI/01/0145/FEDER/007265; FCT/MEC,
food webs, presenting risks to higher trophic levels Fundação para a Ciência e Tecnologia and Ministério da
and to the long-term sustainability of populations, Educação e Ciência, under the Partnership Agreement
with disturbance of the ecosystem balance and PT2020 UID/QUI/50006/2013; SFRH/BPD/86898/
2012). The authors thank Andreia Peixoto for the technical
possible biodiversity lost.
assistance with daphnids and algae.

Conclusions Declaration of interest statement

This study emphasizes the importance of under- The authors declare that they have no conflict of interest.
standing the dynamics that might occur in the
environment due to the presence of HAAs, provid-
ing insight into the biological threats for freshwater Funding
crustacean D. magna as a consequence of direct This work received financial support from the European Union
exposure to these pollutants. Data from compara- (FEDER funds POCI/01/0145/FEDER/007265) and National
tive studies performed at intra- as well as at inter- Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and
lab levels revealed that neonates exhibit an adequate Ministério da Educação e Ciência) under the Partnership
Agreement PT2020 UID/QUI/50006/2013. A. Melo wishes to
sensitivity to allow the performance of ecotoxicolo-
thank the Fundação Ciência Tecnologia grant SFRH/BPD/
gical assays. Results demonstrated consistent effects 86898/2012.
of HAAs on daphnids, particularly the monohaloa-
cetic forms, and that in combination might lead to
impairments that are not predicted by single sub- References
stance studies. The present study also demonstrates
that the application of CA and IA approaches to Acharya, S., K. Mehta, S. Rodrigues, J. Pereira, S. Krishnan,
and C. V. Rao. 1995. Administration of subtoxic doses of
binary and ternary mixtures confirms the high pre- t-butyl alcohol and trichloroacetic-acid to male wistar rats
dictive power of these ecotoxicological theoretical to study the interactive toxicity. Toxicol. Lett. 80:97–104.
models. doi:10.1016/0378-4274(95)03340-Q.
 JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 11

ASTM. 2004. Standard guide for conducting Daphnia magna EPA. 1988. Research and development: Health and environ-
life-cycle toxicity test. Pennsylvania, USA: American mental effects document for chloroacetic acid. Cincinnati:
Society for Testing and Materials. Environmental Protection Agency.
Baird, D. J., A. M. V. M. Soares, A. Girling, I. Barber, EPA. 2002. Methods for measuring the acute toxicity of efflu-
M. Bradley, and P. C. Calow 1989. The long-term main- ents and receiving waters to freshwater and marine organ-
tenance of Daphnia magna for use in ecotoxicity tests: isms 5th ed. Washington, DC: Environmental Protection
Problems and prospects. Proceedings of the First Agency.
European Conference on Ecotoxicology, Lyngby, Denmark, EPA. 2009. Drinking water contaminant candidate list 3-final,
144–48. vol. 74. Washington, DC: Environmental Protection
Barata, C., B. Campos, C. Rivetti, G. A. LeBlanc, S. Eytcheson, Agency.
S. McKnight, M. Tobor-Kaplon, S. de Vries Buitenweg, Faust, M., R. Altenburger, T. Backhaus, H. Blanck,
S. Choi, J. Choi, et al. 2017. Validation of a W. Boedeker, P. Gramatica, V. Hamer, M. Scholze,
two-generational reproduction test in Daphnia magna: M. Vighi, and L. H. Grimme. 2001. Predicting the joint
An interlaboratory exercise. Sci. Total Environ. algal toxicity of multi-component s-triazine mixtures at
579:1073–83. doi:10.1016/j.scitotenv.2016.11.066. low-effect concentrations of individual toxicants. Aquat.
Belden, J. B., R. J. Gilliom, and M. J. Lydy. 2007. How well Toxicol. 56:13–32. doi:10.1016/S0166-445x(01)00187-4.
can we predict the toxicity of pesticide mixtures to aquatic Fisher, D., L. Yonkos, G. Ziegler, E. Friedel, and D. Burton.
life? Integr. Environ. Assess. Manag. 3:364–72. doi:10.1002/ 2014. Acute and chronic toxicity of selected disinfection
ieam.5630030326. byproducts to Daphnia magna, Cyprinodon variegatus, and
Berg, M., S. R. Muller, J. Muhlemann, A. Wiedmer, and Isochrysis galbana. Water Res. 55:233–44. doi:10.1016/j.
R. P. Schwarzenbach. 2000. Concentrations and mass watres.2014.01.056.
fluxes of chloroacetic acids and trifluoroacetic acid in Giller, S., F. LeCurieux, F. Erb, and D. Marzin. 1997.
rain and natural waters in Switzerland. Environ. Sci. Comparative genotoxicity of halogenated acetic acids
Technol. 34:2675–83. doi:10.1021/es990855f. found in drinking water. Mutagenesis 12:321–28.
Bliss, C. I. 1939. The toxicity of poisons applied jointly. Ann. doi:10.1093/mutage/12.5.321.
Appl. Biol. 26:585–615. doi:10.1111/j.1744-7348.1939. Greco, W. R., G. Bravo, and J. C. Parsons. 1995. The search
tb06990.x. for synergy: A critical review from a response surface
Boorman, G. A., V. Dellarco, J. K. Dunnick, R. E. Chapin, perspective. Pharmacol. Rev. 47:331–85.
S. Hunter, F. Hauchman, H. Gardner, M. Cox, and Hanson, M. L., and K. R. Solomon. 2004. Haloacetic acids in
R. C. Sills. 1999. Drinking water disinfection byproducts: the aquatic environment. Part II: Ecological risk
Review and approach to toxicity evaluation. Environ. assessment. Environ. Pollut. 130:385–401. doi:10.1016/j.
Health Perspect. 107:207–17. doi:10.2307/3434484. envpol.2003.12.017.
Bopp, S. K., A. Kienzler, S. C. Andrea-Nicole Richarz, A. van Herrenfreund, S. L., M. A. Pereira, M. D. Khoury, and
der Linden, N. P. Paini, and A. P. Worth. 2019. Regulatory G. Olson. 1987. The carcinogenicity of trichloroethylene
assessment and risk management of chemical mixtures: and its metabolites, trichloroacetic-acid and dichloroacetic
Challenges and ways forward. Crit. Rev. Toxicol. acid, in mouse-liver. Toxicol. Appl. Pharmacol. 90:183–89.
49:174–89. doi:10.1080/10408444.2019.1579169. doi:10.1016/0041-008x(87)90325-5.
Campos, B., R. Jordao, C. Rivetti, M. F. Lemos, A. M. Soares, Heys, K. A., R. F. Shore, M. G. Pereira, K. C. Jones, and
R. Tauler, and C. Barata. 2016. Two-generational effects of F. L. Martin. 2016. Risk assessment of environmental mixture
contaminants in Daphnia magna: Effects of offspring quality. effects. RSC Adv. 6:47844–57. doi:10.1039/C6RA05406D.
Environ. Toxicol. Chem. 35:1470–77. doi:10.1002/etc.3290. Hong, Y., H. Song, and T. Karanfil. 2013. Formation of
CDC. 1999. Achievements in public health, 1900-1999: haloacetic acids from dissolved organic matter fractions
Control of infectious diseases. Morbidity and Mortality during chloramination. Water Res. 47:1147–55.
Weekly Report 48:621–629 doi:10.1016/j.watres.2012.11.025.
Cedergreen, N., A. M. Christensen, A. Kamper, P. Kudsk, Huang, J., N. Graham, M. R. Templeton, Y. Zhang,
S. K. Mathiassen, J. C. Streibig, and H. Sørensen. 2008. C. Collins, and M. Nieuwenhuijsen. 2008. Evaluation of
A review of independent action compared to concentra- Anabaena flos-aquae as a precursor for trihalomethane
tion addition as reference models for mixtures of com- and haloacetic acid formation. Water Sci. Technol.
pounds with different molecular target sites. Environ. 8:653–62. doi:10.2166/ws.2008.151.
Toxicol. Chem. 27:1621–32. doi:10.1897/07-474.1. IARC. 2004. Some drinking-water disinfectants and contami-
EANLS. 2007. The direct toxicity assessment of aqueous envir- nants, including arsenic. Lyon, France: IARC Monographs
onmental samples using the juvenile Daphnia magna immo- on the Evaluation of Carcinogenic Risks to Humans.
bilisation test, Methods for the examination of waters and IARC. 2013. Some chemicals present in industrial and con-
associated materials. Leicestershire: Standing Committee of sumer products, food and drinking-water. Lyon, France:
Analysts Environment Agency (National Laboratory IARC Monographs on the Evaluation of Carcinogenic
Service). Risks to Humans.
12 A. MELO ET AL.

IARC. 2014. Trichloroethylene, tetrachloroethylene, and some Narotsky, M. G., D. S. Best, A. McDonald, E. A. Godin,
other chlorinated agents, vol. 106. Lyon, France: IARC E. S. Hunter, and J. E. Simmons. 2011. Pregnancy loss
Monographs on the Evaluation of Carcinogenic Risks to and eye malformations in offspring of F344 rats following
Humans. gestational exposure to mixtures of regulated trihalo-
ISO. 2012. Water quality - Determination of the inhibition of methanes and haloacetic acids. Reprod. Toxicol. 31:59–65.
the mobility of Daphnia magna straus (Cladocera, doi:10.1016/j.reprotox.2010.08.002.
Crustacea) — Acute toxicity test, 4th. ISO 6341. Geneva, Narotsky, M. G., D. S. Best, E. H. Rogers, A. McDonald,
Switzerland: International Organization for Y. M. Sey, and J. E. Simmons. 2008. Integrated disinfection
Standardization. by-products mixtures research: Assessment of develop-
Kar, S., and J. Leszczynski. 2019. Exploration of computa- mental toxicity in Sprague-Dawley rats exposed to concen-
tional approaches to predict the toxicity of chemical trates of water disinfected by chlorination and ozonation/
mixtures. Toxics 7:15. doi:10.3390/toxics7010015. postchlorination. J.Toxico. Environ. Health A 71:1216–21.
Kienzler, A., S. K. Bopp, S. van der Linden, E. Berggren, and doi:10.1080/15287390802182623.
A. Worth. 2016. Regulatory assessment of chemical mix- Nichols, H. W. 1979. Growth Media-Freshwater. In
tures: Requirements, current approaches and future Handbook of phycological methods: Culture methods and
perspectives. Regul. Toxicol. Pharm. 80:321–34. growth measurements, ed. J. R. Stein, 7–24. Cambridge,
doi:10.1016/j.yrtph.2016.05.020. UK: Cambridge University Press.
Kim, J., Y. Chung, D. Shin, M. Kim, Y. Lee, Y. Lim, and OECD. 1984. Daphnia sp., acute immobilisation test and
D. Lee. 2003. Chlorination by-products in surface water reproduction test. In OECD GUIDELINE FOR TESTING
treatment process. Desalination 151:1–9. doi:10.1016/ OF CHEMICALS. Paris: Organisation for Economic Co-
S0011-9164(02)00967-0. operation and Development.
Kortenkamp, A., T. Backhaus, and M. Faust. 2009. State of OECD. 2004. Guidelines for testing of chemicals. Test No.
the art report on mixture toxicity: Final Report to the 202: Daphnia sp. acute immobilisation test. In OECD
European Commission under Contract Number 070307/ guidelines for the testing of chemicals/section 2: Effects on
2007/485103/ETU/D.1. 2009. biotic systems. Paris: Organisation for Economic Co-
Krasner, S. W., H. S. Weinberg, S. D. Richardson, S. J. Pastor, operation and Development.
R. Chinn, M. J. Sclimenti, G. D. Onstad, and OECD. 2012. Test No. 211: Daphnia magna reproduction
A. D. Thruston Jr. 2006. Occurrence of a new generation test. In OECD guidelines for the testing of chemicals, section
of disinfection byproducts. Environ. Sci. Technol. 2: Effects on biotic systems. Paris: Organisation for
40:7175–85. doi:10.1021/es060353j. Economic Co-operation and Development.
Krasner, S. W., P. Westerhoff, B. Y. Chen, B. E. Rittmann, Plewa, M. J., Y. Kargalioglu, D. Vankerk, R. A. Minear, and
and G. Amy. 2009. Occurrence of disinfection byproducts E. D. Wagner. 2002. Mammalian cell cytotoxicity and
in United States wastewater treatment plant effluents. genotoxicity analysis of drinking water disinfection
Environ. Sci. Technol. 43:8320–25. doi:10.1021/es901611m. by-products. Environ. Mol. Mutagen. 40:134–42.
Kuhn, R., M. Pattard, K. D. Pernak, and A. Winter. 1989a. doi:10.1002/em.10092.
Results of the harmful effects of water pollutants to Rebhun, M., L. HellerGrossman, and J. Manka. 1997.
Daphnia magna in the 21 day reproduction test. Water Formation of disinfection byproducts during chlorination
Res. 23:501–10. doi:10.1016/0043-1354(89)90142-5. of secondary effluent and renovated water. Water Environ.
Kuhn, R., M. Pattard, K. D. Pernak, and A. Winter. 1989b. Res. 69:1154–62. doi:10.2175/106143097x125902.
Results of the harmful effects of selected water pollutants Richardson, S. D., A. D. Thruston, S. W. Krasner,
(anilines, phenols, aliphatic-compounds) to H. S. Weinberg, R. J. Miltner, K. M. Schenck,
Daphnia-magna. Water Res. 23:495–99. doi:10.1016/0043- M. G. Narotsky, A. B. McKague, and J. E. Simmons.
1354(89)90141-3. 2008. Integrated disinfection by-products mixtures
Loewe, S., and H. Muischnek. 1926. Über research: Comprehensive characterization of water con-
Kombinationswirkungen. Naunyn-Schmiedebergs Archiv centrates prepared from chlorinated and ozonated/post-
Für Experimentelle Pathologie Und Pharmakologie chlorinated drinking water. J.Toxico. Environ. Health A
114:313–26. doi:10.1007/bf01952257. 71:1165–86. doi:10.1080/15287390802182417.
Malliarou, E., C. Collins, N. Graham, and Richardson, S. D., M. J. Plewa, E. D. Wagner, R. Schoeny, and
M. J. Nieuwenhuijsen. 2005. Haloacetic acids in drinking D. M. DeMarini. 2007. Occurrence, genotoxicity, and car-
water in the United Kingdom. Water Res. 39:2722–30. cinogenicity of regulated and emerging disinfection
doi:10.1016/j.watres.2005.04.052. by-products in drinking water: A review and roadmap
Muller, S. R., H. R. Zweifel, D. J. Kinnison, J. A. Jacobsen, for research. Mutat. Res. Rev. Mutat. Res. 636:178–242.
M. A. Meier, M. M. Ulrich, and R. P. Schwarzenbach. doi:10.1016/j.mrrev.2007.09.001.
1996. Occurrence, sources, and fate of trichloroacetic acid Rickert, C. I. B., and O. Schmoll, WHO, Edited by. 2016.
in Swiss waters. Environ. Toxicol. Chem. 15:1470–78. Protecting surface water for health. Identifying, assessing
doi:10.1897/1551-5028. and managing drinking-water quality risks in surface-
 JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 13

water catchments. Geneva, Switzerland: World Health Four-Lab Study: Integrated disinfection by-products mix-
Organization. tures research. J.Toxico. Environ. Health A 71:1125–32.
Roberts, J. F., R. van Egmond, and O. R. Price. 2010. Toxicity doi:10.1080/15287390802181906.
of haloacetic acids to freshwater algae. Ecotoxicol. Environ. Sirivedhin, T., and K. A. Gray. 2005. Comparison of the
Saf. 73:56–61. doi:10.1016/j.ecoenv.2009.09.013. disinfection by-product formation potentials between
Sancho, E., M. J. Villarroel, and M. D. Ferrando. 2016. a wastewater effluent and surface waters. Water Res.
Assessment of chronic effects of tebuconazole on survival, 39:1025–36. doi:10.1016/j.watres.2004.11.031.
reproduction and growth of Daphnia magna after different Sorensen, M. V., I. Heiner, O. Ziemer, and B. Neuhaus. 2007.
exposure times. Ecotoxicol. Environ. Saf. 124:10–17. Tubulideres seminoli gen. et sp nov and Zelinkaderes
doi:10.1016/j.ecoenv.2015.09.034. brightae sp nov (Kinorhyncha, Cyclorhagida) from
Scott, B. F., C. Spencer, C. H. Marvin, D. C. MacTavish, and Florida. Helgol. Mar. Res. 61:247–65. doi:10.1007/s10152-
D. C. G. Muir. 2002. Distribution of haloacetic acids in the 007-0073-8.
water columns of the Laurentian Great Lakes and Lake Syberg, K., A. Elleby, H. Pedersen, N. Cedergreen, and
Malawi. Environ. Sci. Technol. 36:1893–98. doi:10.1021/ V. E. Forbes. 2008. Mixture toxicity of three toxicants
es011156h. with similar and dissimilar modes of action to Daphnia
Scott, B. F., C. Spencer, J. W. Martin, R. Barra, magna. Ecotoxicol. Environ. Saf. 69:428–36. doi:10.1016/j.
H. A. Bootsma, K. C. Jones, A. E. Johnston, and ecoenv.2007.05.010.
D. C. G. Muir. 2005. Comparison of haloacetic acids in WHO. 2004. Trichloroacetic acid in drinking-water. back-
the environment of the northern and southern ground document for development of WHO guidelines for
hemispheres. Environ. Sci. Technol. 39:8664–70. drinking-water quality. Geneva, Switzerland: World Health
doi:10.1021/es0501181. Organization.
Simmons, J. E., S. D. Richardson, L. K. Teuschler, WHO. 2017. Guidelines for drinking-water quality, fourth
R. J. Miltner, T. F. Speth, K. M. Schenck, E. S. Hunter, edition, incorporating the 1st addendum. Geneva,
and G. Rice. 2008. Research issues underlying the Switzerland: World Health Organization.