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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study according to protocol.
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples of all test concentrations were taken on preparation, just before and after changing of the test solution.
Freshly prepared stock solutions were also sampled in some occasions.

At least 30 mL was sampled in each case and transferred into the analytical vials for analysis.
Samples from the actual test replicates were filtered using a 0.45 µm GHP Acrodisc filter to remove algae. Filters were first primed with the relevant solution before use.
At the beginning of the test loss of test substance due to filtering was quantified by comparing analysis results of a filtered and an unfiltered sample.

The samples were stored in the freezer until analysis, or analyzed immediately depending upon available storage space and logistical possibilities during the study. If samples were stored, stability samples were stored with the test samples.

Because 30 mL sample was not all used in one analysis run, the remaining sample not used was stored in the freezer immediately after use. If necessary the analyses could then be repeated.

Stability of the test substance during storage was checked twice (day 7 and 22). Samples were found stable over time in the freezer.
Vehicle:
no
Details on test solutions:
- Preparation of the stock solutions:
The test substance is readily soluble in water. To prepare the stock solutions for every water renewal, on average 0.0214 g (standard deviation: 0.0006 g) of test substance was weighed on an analytical balance and then dissolved directly in approx. 160 mL test medium. The stock solutions were agitated mechanically for at least 20 minutes to completely dissolve the test substance.
The pH of the stock solution was checked and found to be between 7.7 and 7.9 and was not adjusted. After this the stock solution was filled up to 200 mL with test medium.
A fresh stock solution was prepared for each solution change.

- Preparation of the test solutions:
Test solutions were prepared by further dilution of the stock solution with test medium.
The ratio between the two concentrations was 2.

Test vessels were filled directly from the test solution containers immediately after preparation. The solutions were renewed three times a week during the test.

The pH of the test solutions were all between 6 and 9 at refreshment and close to the value of the test medium.
One control containing only test medium was included in the test.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test animals were taken from a Daphnia magna clone 5 stock, (Origin: WIL Research Europe, The Netherlands) cultured in conformity with the relevant SOP. The parent animals were cultured in test medium from the day they were born.
The animals used in the test were less than 24 hours old and were obtained from parent animals reproducing parthenogenically and having an age of 2-4 weeks (having previously produced at least one brood before use). The culture is checked yearly for sensitivity by a reference test with potassium dichromate. The results of this test were within the range given in the relevant guideline.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
215 mg/L CaCO3
Test temperature:
Temperature range observed in fresh and used solutions monitored at the moment of renewal: min. 19.3; max. 20.2 °C.
The observed temperature range which was continuously recorded during the test: 19.2 to 20.7 °C.
pH:
7.8 - 8.9
Dissolved oxygen:
9.0 - 11.8 mg O2/L.
Nominal and measured concentrations:
Nominal: 1.0, 2.0, 4.0, 8.0 and 16.0 mg/L
Measured (TWA): 0.44, 1.0, 2.0 mg/L (not all samlpes of the two highest test concentrations were analyzed, therefore no TWA could be calculated)
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL glass beakers
- Type (delete if not applicable): open
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 3 times a week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 medium
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light
- Light intensity: 15.7 µmol/s/m2

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : reproduction, parent length, weight and mortality

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: 21-day range finding test in M4 medium with no effect up to the highest test concentration of 1 mg/L (nominal).
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.49 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% C.I. 0.37 - 0.57 mg/L (TWA)
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.53 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: parental weight
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.7 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: parental length
Details on results:
- Analytical results:
The concentration of the test substance present in the test vessels and the stock solutions was quantified. See for time weighted average (TWA) concentrations in the test Table 1 below.
The measured concentrations of the fresh solutions were all above 80% of the nominal concentrations, whereas the measured concentrations of the used solutions were well below 80%.
Because the measured concentrations of the used solutions were below 80% of the nominal concentration, TWA test substance concentrations were calculated and used for endpoint calculations.
The stock solutions used to for the test solutions showed a good recovery.

- Reproduction:
Analysis of variance should be performed on the number of living neonates per parent animal alive at the end of the test as well as on the number of living neonates per parent animal that did not die accidentally during the test, according to the latest OECD guideline. Concentration related parent mortality was observed, at least for the three highest test concentrations. In 1.0 mg/L (nom.) three daphnids died whereas in 2.0 mg/L (nom.) only two died, also one control animal died during the test (see Table 2). Because it is not sure how many and which incidents were test concentration related, it was chosen to include all animals of the control, 1.0, 2.0 and 4.0 mg/L (nom.) for endpoint calculations as a worst case. This means that it was assumed that all mortalities were caused by the test substance and were all taken into account.

No significant effect on reproduction compared to the control was observed at 0.44 mg/L (TWA, equal to 1.0 mg/L nom.), therefore the Lowest Observed Effect Concentration (LOEC) is 1.0 mg/L (TWA, equal to 2.0 mg/L nom.) and No Observed Effect Concentration (NOEC) is 0.44 mg/L (TWA) for reproduction. EC10- value was 0.49 mg/L (TWA), with 95% confidence limits of 0.37 to 0.57 mg/L (TWA).

- Length and Weight:
Both length and weight data showed no significant differences from the control at 0.44 mg/L (TWA, equal to 1.0 mg/L nom.), therefore NOEC based on length and weight is 0.44 mg/L (TWA).
For length the EC10- value was 0.70 mg/L (TWA) and for weight 0.53 mg/L (TWA).

- Parent mortality:
One parent animal died during the test in the control. Three animals died in 1.0 mg/L, two died in 2.0 mg/L, 9 died in 4.0 mg/L and all animals died in 8.0 and 16.0 mg/L.
An EC50 based on survival of parent animals at the end of the test was calculated to be 1.38 mg/L (TWA).

- Other biological effects observed:
The first brood was in almost all cases released between day 8 and 9 for all test concentrations that produced offspring and the control.
Dead neonates were observed during the test in all replicates of 2.0 and 4.0 mg/L (nom.). Three dead neonates were observed at the end of the test in two replicates of 1.0 mg/L (nom.).
Aborted dead juveniles were observed once in four replicates of 4.0 mg/L (nom.) and once in one replicate of 2.0 mg/L (nom.).

Results with reference substance (positive control):
The culture is checked yearly for sensitivity by a reference test with potassium dichromate. The results of this test were within the range given in the relevant guideline.
Reported statistics and error estimates:
The data on reproduction, parental length and weight were tested for normality using Shapiro Wilk’s test, Bartlett’s tests was used for homogeneity of variance.
Analysis of variance was performed on the number of living neonates per parent animal which did not die accidentally during the test, and parental length using Williams’ test. For parental weight data Bonferroni t test was used.
EC10- and EC50- values were determined for reproduction and length using Maximum Likelihood Probit analysis. In order to determine an EC10- value for weight linear interpolation was used. For parental mortality Trimmed Spearman-Karber was used to determine the EC50.

Toxcalc was used for all computations.
Validity criteria fulfilled:
yes
Conclusions:
This study is reliable without restrictions. It is performed according to OECD guideline 211 under GLP and all critical validity criteria were fulfilled.
The calculated endpoints are adequate for C&L and risk assessment purposes.
Executive summary:

The purpose of this study was to assess the toxicity of the test substance dissolved in fresh water, on the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a semi-static test complying with the OECD Guideline No. 211.

The test criterion of toxicity used was reproductive capacity expressed as the number of neonates per daphnid during the study.

 

The nominal concentrations used in the study were as follows: 0, 1.0, 2.0, 4.0, 8.0and 16.0 mg/L

All concentrations given refer to the technical product as supplied by the sponsor.

 

Analytical determinations of the relevant test solutions were made on all fresh and used solutions during the test.

The results of the chemical analyses show that the concentrations in the test solutions freshly prepared were close to nominal test concentrations whereas test solutions used were below 80% of the nominal concentration. Therefore, time weighted average (TWA) test substance concentrations were determined and used to calculate the effects.

 

The validity criteria were respected:

Cultures were in good health (i.e. disease free, no ephippia or males, no discolored animals).

One parent animal died in the control group over the test period, which is less than 20%.

The average number of juveniles per parent animal alive at the end of the test in the control was 144 after 21 days.

 

The No Observed Effect Concentration (NOEC) is determined as the concentration used in the study that is immediately below the Lowest Observed Effect Concentration (LOEC), the latter derived statistically from the data using the appropriate statistical test.

EC10-and EC50-values were determined where possible, using statistical analysis.

 

No significant effects were found up to 0.44 mg/L (TWA, equal to 1.0 mg/L nom.) on reproduction and parental length and weight. NOEC for these endpoints is equal to 0.44 mg/L (TWA).

The lowest EC10was found for reproduction and was 0.49 mg/L (TWA).

Description of key information

The lowest 21d-EC10 was found for reproduction and was 0.49 mg/L (TWA).

Key value for chemical safety assessment

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

Additional information

The chronic toxicity of the test item tert-Butyl peroxybenzoate to Daphnia magna was determined in a 21 -day semi-static test according to OECD guideline 211.

The results of the chemical analyses show that the concentrations in the test solutions freshly prepared were close to nominal test concentrations whereas test solutions used were below 80% of the nominal concentration. Therefore, time weighted average (TWA) test substance concentrations were determined and used to calculate the effects.

No significant effects were found up to 0.44 mg/L (TWA, equal to 1.0 mg/L nom.) on reproduction and parental length and weight. NOEC for these endpoints is equal to 0.44 mg/L (TWA).

The lowest EC10was found for reproduction and was 0.49 mg/L (TWA).