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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-03-02 to 2020-10-20 (Exposure period: 2020-03-04 to 2020-03-25)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study with analytical test item concentration verification
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
DETAILS OF TEST MATERIAL
- lot/batch number of test material: 18315014
- Purity: 97.0 corr. area-%
- Physical state: liquid
- Color: colorless, clear
- Density: 0.9927 g/mL (BASF, see IUCLID Ch. 4.4)
- Vapor pressure: p/hPa = 2.2E-06 (20°C); 4.2E-06 (25°C); 8.3E-05 (50°C) (BASF, see IUCLID Ch. 4.6)
- Water solubility: 32 g/L ± 1 g/L at 20°C ± 0.5 °C (BASF, see IUCLID Ch. 4.8)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: ambient (room temperature)
- Storage stability of test material: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability of the test substance as a solution in test water and under testing conditions was determined by concentration control analysis. The test solutions were visibly homogeneous.

OTHER SPECIFICS
- adjustment of pH: no
Analytical monitoring:
yes
Details on sampling:
- Sampling method: The concentration of the test substance was determined in one representative test interval per week in each freshly prepared test solution (test vessels without daphnids) and in the 48 h or 72 h old test solutions before renewal (combined sample from test vessels with daphnids).
- Exactly 10 mL of each sample was stabilized with 10 μL formic acid for analytical monitoring.
Vehicle:
no
Details on test solutions:
PREPARATION OF STOCK SOLUTION AND TEST SOLUTIONS
- The stock test solution (11.3 mg/L) was prepared by dissolving approx. 22.6 mg of test substance in 2 L of test medium and stirring for approximately 20 min on a magnetic stirring plate.
- The stock solutions appeared colorless-clear.
- The lower test concentrations were prepared by dilution of this stock solution
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: The clone of Daphnia magna STRAUS 1820 used was supplied by the Institut National de Recherche Chimique Appliquée, France, in 1978. From this date on this clone was cultured and bred continuously in the investigating laboratory.
- Age of parental stock (mean and range, SD): Daphnia brood stock are kept in mass cultures consisting of approx. 20–30 individuals for a maximum of 4 weeks. All individuals in the mass culture originate from a single female. After approximately 14 days the adults have produced at least 3 broods and the young can be used in tests.
- Age at start of exposure: <24 h
- Feeding during test: Yes
- Food type: During the test daphnids were fed daily a diet of live green algae Desmodesmus subspicatus, cultured in a synthetic medium.
- Amount: The daphnids were fed a defined volume (≤114 μL) of the concentrate to reach the respective amount of food determined as total organic carbon (mg TOC) (Day 0-3: 0.1 mg TOC, day 4-5: 0.12 mg TOC, day 6-7: 0.14 mg TOC, day 8-test end: 0.2 mg TOC). The algae were stored in a refrigerator (dark, about 4-8°C) for maximum 21 days.
- Frequency: daily

ACCLIMATION
- Acclimation period: The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4, water quality, temperature (20 ±1°C), and diet.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
- Control: 2.29 to 2.40 mmol/L
- Treatments: 2.3 to 2.43 mmol/L
Test temperature:
- Control and Treatments: 20-21°C.
pH:
- Control: 7.7 - 8.1
- Treatments: 7.7 - 8.4
Dissolved oxygen:
- Control: 8.4- 9.1 mg/L
- Treatments: 8.3 - 9.6 mg/L
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 0.11, 0.34, 1.1, 3.5, 11.3 mg/L as nominal
concentrations based on test substance mass without correction for purity.

- Measured concentrations (overall mean): 0.12, 0.34, 1.12, 3.55, 11.35 mg/L.
All measured concentrations deviated less than 20% from the nominal concentration besides one sample (124%). Thus, the test substance was stable in solution under test conditions and nominal concentrations are an accurate representation of exposure levels maintained throughout the test period. Following recommendations in OECD 211 the results should be evaluated based on the nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Numbered glass beakers (nominal volume 100 mL), covered with glass Petri plates to slow evaporation.
- Aeration: none
- Type of flow-through: semi-static
- Renewal rate of test solution: 3 times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 0.02 animals / mL

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: A synthetic fresh water (Elendt M4) is used as media for culture and test purposes. For the composition of this M4 medium see OECD 211
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16:8 light/dark
- Light intensity: 696–782 lux at a wave length of 400–750 nm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Adult mortality: daily
- Number of juveniles: daily
- Stillborn or immobile juveniles and aborted eggs: daily
- growth: test end
Reference substance (positive control):
yes
Remarks:
Sodium chloride
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 11.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
analytically verified
Basis for effect:
mortality
Remarks on result:
other: The data were not sufficient to calculate NOEC or LOEC. Effect greater than the highest concentration with data for the endpoin
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
3.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
analytically verified
Basis for effect:
reproduction
Remarks on result:
other: not pH adjusted
Details on results:
- Mortality of parent animals was observed in 4 treatment groups; however it was not statistically significant and did not follow a concentration response pattern (negative Cochran-Armitage test). The observed mortalities were either accidental or inadvertent.
- The reproduction endpoint is based on offspring per surviving adult, since there was no concentration response pattern to the mortality.
- There was no significant effect on Daphnia reproduction in test groups with nominal concentrations >=3.5 mg/L.
- Significant effects on reproduction were observed at 11.3 mg/L. Organisms in the highest test group had a 95% reduction in reproduction.

- Since the measured concentrations of the test substance in the test solutions were generally within +20% of the nominal concentrations, the effect concentration can be expressed relative to the nominal concentration.
Results with reference substance (positive control):
Reference substance testing:
The EC50(48 h) of the reference substance sodium chloride (NaCl) was 5.13 g/L (experiment date: 25 Mar 2020).
This result is within the range of 3.88–7.22 g/L, which represents ±2 standard deviations from the published EC50 (48 h) of 5.55 g/L and indicates that the culture of Daphnia magna used in this study is responding normally to toxic stress.
Reported statistics and error estimates:
The following parameters and statistical procedures (hypothesis tests) were
used to evaluate significant differences to the control:
- Reproduction as number of living young (Wilcoxon’s test, one sided with Bonferroni Holm adjustment, one sided analysis)
- Parent mortality as immobilized organisms (Fishers exact test, one sided analysis)
- % immobile young (Wilcoxon’s test, with Bonferroni Holm adjustment, one sided analysis)
- % aborted eggs (Wilcoxon’s test, one sided with Bonferroni Holm adjustment, one sided analysis)
- Time to first brood (Wilcoxon’s test, with Bonferroni Holm adjustment, one sided analysis)
- Growth as length in mm (Dunnett´s test, one sided analysis)

Preview of Extracted Tabular Data


 


Table: Analytically measured concentrations of the test substance in the test solutions








































Nominal concentration [mg/L]Mean measured concentration
[mg/L]a
% of nominal
0 (control)* 
0.110.12111
0.340.34100
1.11.12102
3.53.55101
11.311.35100

a: based on 6 measured renewal period days;
*: <LOQ and 14 μg/L detected in control samples


 


Table: Mortality and Reproduction Summary after 21 days.


























































Test Groups
Nominal
concentration
[mg/L]
ReproductionMortality
Mean Living
Young
per surviving
adult
% effectbParent animals% effectb
0 (Control)125 (11.6%a)-0-
0.11135-1-
0.34134-1-
1.1142-0-
3.5126-1-
11.36**95%2-

*: p≤ 0.05; **: p≤ 0.01
a: Coefficient of variation for control fecundity based on surviving parent; has to be ≤25%.
b: Effect relative to control. Only calculated for statistically significant effects.


 


Table: Other biological observations among surviving parent animals in each concentration after a 21 day exposure






















































Test Groups
Nominal
concentration
[mg/L]
Mean Growth
(length, mm)
% Immobile
Young
Mean Days to
First Brood
% Aborted Eggs
0 (Control)4.309.40.4
0.114.309.10.6
0.344.20.29.30.1
1.14.40.390.1
3.54.31.1**9.20.5
11.32.7**3.513**1.6

*: p≤ 0.05; **: p≤ 0.01


 


Table: Summarized effect parameters for the main endpoints affer 21 days based on nominal concentrations














































EndpointEffect Concentrations [mg/L]
NOECLOEC
Reproduction3.511 .3
Mortality≥11.3[a]≥11.3[a]
Growth (Length)3.511.3
Time to first brood3.511.3
Aborted Eggs≥11.3[a]≥11.3[a]
Immobile Young≥11.3[a]≥11.3[a]
[a] The data were not sufficient to calculate NOEC or LOEC. Effect greater than the
highest concentration with data for the endpoint.

 


Table: Measured values for the oxygen content [mg/L], measured in replicate 1 per test concentration.







































































Nominal concentrations [mg/L]Day of the test
0a2b9a12b19a21b
09.08.98.89.18.88.4
0.119.08.98.89.08.88.5
0.349.08.98.88.98.88.6
1.19.18.98.88.98.88.4
3.59.08.98.89.08.88.3
11.39.08.98.99.68.99.6

a: in the freshly prepared test solutions, b: before renewal in the old test solutions


 


Table: pH-values, measured in replicate 1 per test concentration







































































Nominal concentrations [mg/L]Day of the test
0a2b9a12b19a21b
07.88.17.97.87.87.7
0.117.88.17.97.87.87.7
0.347.88.17.87.77.87.7
1.17.88.17.97.77.87.7
3.57.88.17.97.77.97.7
11.38.08.18.18.18.08.4

a: in the freshly prepared test solutions, b: before renewal in the old test solutions


 


Table: Instantaneous temperature (ºC), measured in replicate 1 per test concentration.







































































Nominal concentrations [mg/L]Day of the test
0a2b9a12b19a21b
0202120212121
0.11202120212021
0.34202020212021
1.1202020212021
3.5202020212021
11.3202021212121

a: in the freshly prepared test solutions, b: before renewal in the old test solutions


 


Table: Measured values for the total hardness [mmol/L]







































































Nominal concentrations [mg/L]Day of the test
0a2b9a12b19a21b
02.292.322.322.362.382.40
0.112.302.342.342.372.382.43
0.342.312.322.322.382.382.41
1.12.312.332.332.372.382.42
3.52.302.332.322.362.372.41
11.32.342.342.332.372.382.43

a: in the freshly prepared test solutions, b: before renewal in the old test solutions


 


 


This test was fully compliant with all the following validity criteria required by the corresponding test guidelines and is considered valid.
- In the control the mortality of parent animals up to the end of the test should be ≤20%.
- In the control the mean number of living young per parent animal that survived the test should be ≥60.

Validity criteria fulfilled:
yes

Description of key information

Based on the GHS, no chronic toxicity is anticipated at an NOEC or EC10 > 1 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
NOEC
Remarks:
Reproduction
Effect concentration:
3.5 mg/L

Additional information

The long-term toxicity to aquatic invertebrates was assessed under semi-static conditions in an OECD 211 study using Daphnia magna as test organism (BASF, 2020, Report no. 51E0241/13E078).


Based on the reproduction the following effect value was determined: 21-d NOEC = 3.5 mg/L (based on nominal concentrations, verified by analytical measurement).


The pH of the test solutions were not adjusted and all validity criteria according to the OECD guideline were fullfilled.


With high probability the substance is concluded to be chronically not harmful to aquatic invertebrates.