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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:
30.09. to 23.10.2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
Adopted: October 2, 2012
Deviations:
yes
Remarks:
with no impact on proper study performance and study results (see Any other information)
Qualifier:
according to
Guideline:
EU Method C.20 (Daphnia magna Reproduction Test)
Version / remarks:
Council Regulation (EU) No. 2017/735, published in O.J. L 112, 2017
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Because the test item is poorly soluble, the test solutions (1, 5 and 10 mg·L-1) were prepared by direct weighing the test item into a graduated flask. The test solutions were ultrasonicated for 30 minutes and then were stirred for 1 hours. After the period of stirring the test solutions were filtered through 0.45 μm filter and these solutions were used for testing.
The stock solutions of the M4 medium were prepared by adding measured amounts of the individual chemicals to deionized water.
The concentration of the test item in M4 medium were prepared three times a week for the preliminary test. The frequency of test solutions renewal was three times per week, without aeration. Fresh test solutions were prepared and parent animals were transferred to them by glass pipette.
Control test was performed concurrently with M4 medium only. The volume of the test solutions was 100 mL in each test beaker. 10 pieces of daphnia were put individually into each test concentration and control series.

Preliminary test: 10, 5, 1, 0 mg·L-1
Definitive test: Based on no toxicity of the test item found in the preliminary test, the study was terminated.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
Daphnia magna Straus

- Source, method: aperiodic parthenogenesis from own laboratory breeding
The grow-up gravid females are separated into M4 medium. Young daphnia born in 24 hours are segregated by observation under a microscope. Only the females are transferred to the test containers.

- Feeding during test, type, amount, frequency: daily (out of 05.10., 06.10., 12.10., 13.10., 19.10., 20.10.), the mixture of two algae (Desmodesmus subspicatus and Raphidocelis subcapitata 1:1), ration levels 0.5 mg C/daphnia/day at all times

Volume of the mixture of algae for feeding: 477 µL/100 mL in preliminary test
Test type:
semi-static
Water media type:
freshwater
Remarks:
The stock solutions of the M4 medium were prepared by adding measured amounts of the individual chemicals to deionized water.
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
none
Hardness:
above 2.4 mmol·L-1
Preliminary test: 2.42 – 2.47 mmol Ca+Mg /L
Test temperature:
20 ± 2°C, with a variation of ± 2°C at each individual test
pH:
6 – 9 and should not change more than 1.5 pH unit
Preliminary test: 8.1 - 8.5
Dissolved oxygen:
higher than 3 mg·L-1
Preliminary test: 7.2 – 7.9
Salinity:
n.a.
Conductivity:
Preliminary test: 1.39 – 2.73 µS·cm-1
Nominal and measured concentrations:
Preliminary test: 10, 5, 1 mg·L-1
Details on test conditions:
TEST SYSTEM
- Material, size, headspace, fill volume: 100 ml
- Aeration: without
- Renewal rate of test solution (frequency/flow rate): 3× per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Volume of the mixture of algae for feeding: 477 µL/100 mL in preliminary test

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water
- Test medium: M4
- Conductivity of the deionized water: preliminary test: 1.39 – 2.73 µS·cm-1

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light
- Light intensity: 1568 – 1575 lx (not exceeding 1250 – 1670 lx in general)
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
The following data were recorded during the test: numbers of living offspring by each parent animal, presence of dead offspring and mortality parent animals. The total number of offspring per parent animal was calculated for each test vessel. If in any test vessel the parent animal died during the test, then the test vessel was excluded from the analysis.
The mean reproductive output for each concentration and control were calculated. Reduction of reproductive output was expressed in %.

- Mortality of control: 0 parent animal; 0 offspring


Results with reference substance (positive control):
a) Reference test
The sensitivity of the test species and correctness of test performance is periodically verified by testing with the reference substance, potassium dichromate. The results of the verification test with K2Cr2O7 are the following:
21 days – EC50 = 0.26 mg/L (95% confidence limit: 0.25 – 0.27 mg/L)

b) Data in the literature
HERMENS, JOOP, et al. Joint effects of a mixture of 14 chemicals on mortality and inhibition of reproduction of Daphnia magna. Aquatic Toxicology, Volume 5, Issue 4, November 1984, Pages 315-322.
21 days – EC50 = 0.27 mg/L

c) Method validity evaluation
The reduction in reproductive output of the test organism by the reference substance (21d – EC50) obtained from our reference test is conformable to the values stated in the literature (Hermens et al., 1984).

The total number of living and dead daphnia

Concentration

mg·L-1

Living offspring

Total

Living offspring

of survived

parents

Dead

parent animals

Dead offspring

Total

10

597

597

0

0

5

579

579

0

0

1

593

593

0

0

K

606

606

0

0

The evaluation of reduction in reproductive output of daphnia

Concentration

mg·L-1

The mean number
of the living offspring/daphnia

Reduction in reproductive output versus control

%

10

59.7

1.5 %

5

57.9

4.5 %

1

59.3

2.1 %

K

60.6

-

Detailed information on offspring per individual replicate or concentration-response curve are provided by file in section Attached background material.

Validity criteria fulfilled:
yes
Remarks:
see Overall remarks
Conclusions:
The observed reduction in reproductive output of Daphnia was 1.5 % in the highest concentration in the preliminary test. Therefore exact values of EC50 could not be calculated and the values of EC are given in the form of a range. The nominal concentrations of the test item were used for all evaluations and results.

Preliminary test showed that it could be reasonable to expect that estimated EC50 will be higher than 10 mg·L-1:
21 days – EC50 > 10 mg/L

In congruence with study plan, when the assumed value of EC50 is higher than 10 mg·L-1, no further testing is needed and the result obtained in preliminary test presents the result of whole study.
Definitive test was not needed, because no effect was observed at the highest concentration in the preliminary test and arrangement of preliminary test meets conditions specified in guideline for limit test (ten replicates for both the treatment and control groups and the timelines is 21 days).
Executive summary:

The test item, Pigment Red 4, was tested in reproduction test on Daphnia magna.

The test was performed according OECD Test Guideline No. 211 - Daphnia magna Reproduction Test. Adopted: October 2, 2012.

The test item is poorly soluble substance, the test solutions were prepared by direct weighing the test item into a graduated flask with M4 medium. The test solutions were ultrasonicated for 30 minutes and then were stirred for 1 hours. After the period of stirring the test solutions were filtered through 0.45 μm filter.

The preliminary test was performed in a range of the test item concentration 1 – 10 mg·L-1. The test was performed as semi-static, frequency of the test solutions renewal was three times a week.

The calculated reduction in reproductive output was 1.5 % in the highest concentration, the exact values of EC50 could not be calculated on the basis of data acquired. Test result is given in the form of a range. In congruence with study plan, when the assumed value of EC50 is higher than 10 mg·L-1, no further testing is needed and the result obtained in preliminary test presents the result of whole study.

The experiment with the concentration of 10 mg·L-1 included in the preliminary test has been performed using ten replicates for both the treatment and the control groups, therethrough the conditions of the limit test given in guideline have been fulfilled and no further testing was needed.

The study was performed without analytical determination of the test item concentrations. The suitable analytical method for the determination of the test item concentration was not available.

For all evaluations and results the nominal concentrations of the test item were used.

Test results, based on nominal concentration:

21 days – EC50 > 10 mg/L

Description of key information

The observed reduction in reproductive output of Daphnia was 1.5 % in the highest concentration in the preliminary test. Therefore exact values of EC50 could not be calculated and the values of EC are given in the form of a range. The nominal concentrations of the test item were used for all evaluations and results.

Preliminary test showed that it could be reasonable to expect that estimated EC50 will be higher than 10 mg·L-1:

21 days – EC50 > 10 mg/L

 

In congruence with study plan, when the assumed value of EC50 is higher than 10 mg·L-1, no further testing is needed and the result obtained in preliminary test presents the result of whole study.

Definitive test was not needed, because no effect was observed at the highest concentration in the preliminary test and arrangement of preliminary test meets conditions specified in guideline for limit test (ten replicates for both the treatment and control groups and the timelines is 21 days).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
10 mg/L

Additional information