tert-butyl 3,5,5-trimethylperoxyhexanoate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-03-20 until 2014-04-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

02 October 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Since the concentration of the test item was not expected to remain within ± 20 per cent of the nominal before renewal, as shown during analytical method validation analysis was performed from each testing concentration at the start and at the end of each water renewal period. Controls were analysed at the start of each water renewal period.

Vehicle:

yes

Details on test solutions:

The test solutions used in the test were prepared by mechanical dispersion using acetone as water-miscible organic solvent and then diluted with Elendt M4 medium. The test solutions were freshly prepared at the beginning of the experiment and subsequently at the start of each water renewal period in the testing laboratory.
The test item is poorly water soluble material. With respect to the difficult separation of the non-dissolved part of the test substance, the test solutions were prepared using water-miscible solvent (acetone) in order to obtain constant initial concentrations at each water renewal period and thus minimise the variability of the individual concentrations during the exposure period. The test concentrations were below the solubility level of the test item in the test medium in each case.
For preparation of the test concentrations, individual stock solutions were first prepared by dissolving an appropriate amount of test item in acetone. The test solutions of the chosen test concentrations were prepared by appropriate diluting of these stock solutions in the dilution water (Elendt M4 medium) using approx. 5 min stirring. The concentration of the solvent (acetone) was 0.1 mL/L in each test concentration and in the additional solvent control.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Source: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
- Age of parental stock: less than 24 hours at the start of the test
- Feeding during test: The Daphnia were fed daily and the supplied diet was based on the amount of organic carbon (C) provided to each parent animal. This amount was 0.15 mg C/Daphnia/day

ACCLIMATION
The stock animals were maintained in same culture conditions (light, temperature, medium, feeding etc.) as that used during the exposure period, therefore additional acclimatisation to avoid stressing the parent animals prior to test was not necessary. Parent animals used in the test were not the first brood progeny

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

No

Hardness:

The measured hardness values were in the range of 201 – 231 mg/L (> 140 mg/L) expressed in CaCO3

Test temperature:

The measured values were in the range of 18 – 22 °C and did not vary by more than 2 °C

pH:

7.64 – 8.98 (the pH remained within the range of 6 – 9)

Dissolved oxygen:

6.46 – 9.23 mg/L (all measured value were > 3 mg/L)

Nominal and measured concentrations:

Nominal test concentrations: 0.19, 0.38, 0.75, 1.50, 3.00 mg/L.
Measured concentrtaions: 0.12, 0.22, 0.44, 0.89, 1.65 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sealed glass beakers (50 mL volume) with approx. 50 mL test medium
- Type: closed
- Aeration: not aerated
- Renewal rate of test solution: this reproduction test was carried out in a semi-static renewal system. Based on the results of the analytical method validation study the test item is not stable in the test medium (Elendt M4 medium) for at least two days in lower concentrations (i.e. below 80 % of initial concentration), therefore the frequency of test solution renewal was daily.
When the medium was renewed a second series of test vessels was prepared and the parent animals transferred to them by a pipette of suitable diameter. The volume of test medium transferred with the Daphnia was minimized.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
Elendt M4 Medium (during the breeding and during the test)
The Elendt M4 Medium (according to OECD 211) was prepared as follows:
Separate stock solutions of individual trace elements were first prepared in deionised water. From these different stock solutions a second single stock solution was prepared, which contained all trace elements (combined solution).

OTHER TEST CONDITIONS
- Adjustment of pH: The test was carried out without adjustment of pH
- Photoperiod: 16 h light : 8 h dark
- Light intensity: The measured light intensity values were in the range of 15-20 µE m-2s-1

EFFECT PARAMETERS MEASURED:
The mortality among the parent animal was recorded daily. The length of the parent animals (i.e. body length excluding the anal spine) was measured at the end of the test. The length of the parent animals (i.e. body length excluding the anal spine) was measured at the end of the test. Other parameters: including time to production of first brood (and subsequent broods), number of broods per surviving parent animal, number of aborted broods were noticed.
(The temperature, oxygen concentration and pH values were measured at least once a week in fresh and old media, in the controls and in the highest test item concentration. In addition, the temperature was continuously monitored (with a min/max thermometer) within the climate chamber during the experimental period. Measurement of hardness was performed at least in each batch of reconstituted water prepared for the experiment. The light intensity was confirmed at the start and at the end of the test within the climate chamber).

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 1, 2, 5 mg/L
- Results used to determine the conditions for the definitive study:
By nominal concentrations of 0, 1, 2, 5 mg/L the following results (number of immobilised animals at 48 h) were obtained respectively: 0/10 (daphnis), 0/10, 0/10 and 8/10.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.44 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.58 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % confidence limits: 0.43 – 2.66 mg/L

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.33 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % confidence limits: 0.28 – 0.36 mg/L

Details on results:

Mortality of parents:
The mortality among the parent animal was recorded daily.
Twenty per cent mortality was observed for parent animals of the untreated control and 10 % mortality was observed in the solvent control group. The occurred mortality was similar in the concentrations of 0.12 and 0.22 mg/L (20 and 10 % respectively).
Statistically significantly different mortality (70 %) was observed in the two highest concentrations (20 and 10 % respectively). Somewhat higher mortality (40 %) was observed in the 0.44 mg/L concentration, however the statistical evaluation (Bonferroni t-Test, α=0.05) showed no significant difference compared to the solvent control. Statistically significantly different mortality (70 %) was observed in the two highest concentrations (0.89 and 1.65 mg/L; measured) during the test and was considered to be due to test item effect.

Offspring:
The offspring produced by each parent animal were removed and counted daily from the appearance of the first brood.
The mean number of living offspring produced by parent animals having survived at the end of the test was 90.25 in the untreated control and 90.67 in the solvent control group. Similar or slightly higher numbers (104.38 and 90.89) were obtained for two lowest test concentration (0.12 and 0.22 mg/L; measured). Lower and statistically significantly different values compared to the solvent control (according to Bonferroni t-Test, α=0.05) was noticed in the concentrations of 0.44, 0.89 and 1.65 mg/L (measured).
Based on the results of statistical evaluation (Bonferroni t-Test, α=0.05) with parent mortality data, replicates of dead parents were excluded from reproductive performance analysis in the three lowest concentration and controls (where the parental mortality did not follow a concentration-response pattern). Parent mortality was considered to be due to test item effect in the two highest concentrations and therefore replicates of dead parents were included in the data analysis related to reproduction.
The 21-day NOEC related to reproduction was determined to be 0.22 mg/L (measured). The 21-day LOEC was 0.44 mg/L (measured).
The ECx values of the test item and their confidence limits related to reproduction were calculated using Probit analysis by TOXSTAT software.
The 21-day EC50 was determined to be 0.58 mg/L (95 % confidence limits: 0.53 – 0.62 mg/L). The 21-day EC10 was determined to be 0.33 mg/L (95 % confidence limits: 0.28 – 0.36 mg/L).

Growth Measurement:
At the end of the test body length measurement was performed and the obtained data were statistically evaluated by analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) using TOXSTAT software.
Statistically significant difference compared to the solvent control group was noticed in the two highest tested concentrations of 0.89 and 1.65 mg/L (measured).

Other Parameters:
Aborted broods were observed in some cases at the two highest concentration (0.89 and 1.65 mg/L; measured).
Aborted broods (AB) observed during the test:
Concentration 0.89 mg/L:
– 2 AB in replicate 7 on 7th day
– 6 AB in replicate 9 on 7th day
Concentration 1.65 mg/L:
– 5 AB in replicate 7 on 11th day
– 8 AB in replicate 10 on 11th day
– 6 AB in replicate 7 on 19th day
– 4 AB in replicate 10 on 21st day
The total number of aborted broods counted during the experiment was 8 in the 0.89 mg/L concentration and 23 in the 1.65 mg/L concentration.
Stumpy limbs of parent animals were noticed in the highest test concentration (1.65 mg/L; measured) at the end of the study (on 21st day).
Presence of male neonates or ephippia was not noticed in this test.

Reported statistics and error estimates:

For the estimation of LOEC and NOEC values for effects of the test item on the reproductive output of Daphnia the mean value and standard deviation for reproductive output of each concentration was calculated and results were analysed by analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) using TOXSTAT software compared with the Solvent Control. The data were checked for homogeneity of variance and normality.
In order to consider whether or not the parental mortality in exposed replicates followed a concentration-response pattern mortality data were analysed by analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) using TOXSTAT software.
Statistical analysis was assessed with the additional determined body length values by analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) using TOXSTAT software compared with the Solvent Control.
The concentration which caused x per cent reduction in reproductive output (i.e. ECx value) was calculated by Probit analysis with 95 % confidence limits using the TOXSTAT software.

Validity criteria fulfilled:

yes

Conclusions:

In the 21-day semi-static chronic toxicity test to Daphnia magna with the tests substance the 21-day NOEC value based on reproduction was determined to be 0.22 mg/L. The 21-day LOEC based on reproduction was determined to be 0.44 mg/L. The 21-day EC50 value based on reproduction was determined to be 0.58 mg/L. The 21-day EC10 value based on reproduction was determined to be 0.33 mg/L. The results are based on the measured concentrations.

Executive summary:

The purpose of this study was to evaluate the influence of the test item on the reproductive output of Daphnia magna in a semi-static test water renewal system, with daily renewal periods. Young female Daphnia (the parent animals) aged less than 24 hours at the start of the test were exposed to aqueous test media (Elendt M4 Medium) containing the test item for 21 days at a range of concentrations. The nominal test item concentrations were 0.19, 0.38, 0.75, 1.50 and 3.00 mg/L. The measured test item concentrations deviated more than 20 % from the nominal during the experiment therefore the time-weighted mean of the measured start and end concentrations at each water renewal period were calculated in order to determine exposure concentrations. The calculated time-weighted mean concentrations were the followings: 0.12, 0.22, 0.44, 0.89 and 1.65 mg/L. All biological results are based on these measured test item concentrations. The test solutions were prepared using water-miscible solvent (acetone). An untreated control and an additional solvent control group were investigated concurrently. The concentration of the solvent (acetone) was 0.1 mL/L in each test concentration and in the additional solvent control. The animals were held individually (ten replicates/treatment) and were fed daily, with diet supplied based on the amount of organic carbon (C) per parent animal (constant 0.15 mg C/Daphnia/day). Analytical method: The quantification of the test item was performed by a previously validated analytical method using HPLC analysis. Samples were taken from each testing concentration at the start and at the end of each water renewal period. Controls were analysed at the start of each water renewal period. The validity criteria of the test were fulfilled. Biological results: The offspring produced by each parent animal were removed and counted daily from the appearance of the first brood. The statistical evaluation of the mean number of living juveniles showed that the offspring production for concentrations 0.44, 0.89 and 1.65 mg/L (measured) was statistically significantly different from that of the solvent control. No significant reduction of the offspring production was observed in the two lowest test concentration (0.12 and 0.22 mg/L; measured) and in the untreated control when compared to the solvent control group. Accordingly, the 21-day NOEC related to reproduction was determined to be 0.22 mg/L (measured). The 21-day LOEC was 0.44 mg/L (measured). The 21-day EC50 was determined to be 0.58 mg/L (95 % confidence limits: 0.28 – 0.36 mg/L). Aborted broods were observed in some cases at the concentrations of 0.89 and 1.65 mg/L (measured). The total number of aborted broods counted during the experiment was 8 in the 0.89 mg/L, 23 in the 1.65 mg/L. Stumpy limbs of parent animals were noticed in the highest test concentration (1.65 mg/L; measured) at the end of the study (on 21st day). The mortality among the parent animals was recorded daily. In order to consider whether or not the parental mortality in exposed replicates followed a concentration-response pattern mortality data were analysed by analysis of variance (ANOVA) and Bonferroni t-Test (α= 0.05). Significant mortality was observed in the two highest concentration (0.89 and 1.65 mg/L; measured) when compared to the solvent control, therefore mortality in these concentrations were considered as a test item effect and the produced number of juveniles in replicates where parental mortality occurred were also included in the data analysis related to reproduction. In the lower concentrations (as well as in the controls) no significant parent mortality was observed (based on the statistical evaluation) and therefore the replicates of dead parents were excluded from reproductive performance analysis. At the end of the test body length measurements of the parent animals were performed and the obtained data was statistically evaluated. Statistically significant differences were observed for concentrations 0.89 and 1.65 mg/L (measured), when compared to the solvent control group.

Description of key information

The following results were obtained with the test substance in a 21-day semi-static chronic toxicity test to Daphnia magna: NOEC value of 0.22 mg/L based on reproduction; LOEC value of 0.44 mg/L based on reproduction and EC50 and EC10 values 0.58 and 0.33 respectively based on reproduction as well. The results were based on measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.22 mg/L

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

0.33 mg/L

Additional information

The study evaluated the effects of the test substance on the reproductive output of Daphnia magna. Young female Daphnia were exposed to aqueous test media containing the test item for 21 days at a range of concentrations.

The nominal test item concentrations were 0.19, 0.38, 0.75, 1.50 and 3.00 mg/L. The measured test item concentrations deviated more than 20 % from the nominal during the experiment therefore the time-weighted mean of the measured start and end concentrations at each water renewal period were calculated in order to determine exposure concentrations. The calculated time-weighted mean concentrations were the followings: 0.12, 0.22, 0.44, 0.89 and 1.65 mg/L. All biological results are based on these measured test item concentrations.

The test was run in a semi-static water renewal system, with daily renewal periods. The test solutions were prepared using water-miscible solvent (acetone). An untreated control (only Elendt M4 Medium) and an additional solvent control group were investigated concurrently. Ten Daphnia were investigated in each group (per concentration and controls). Samples were taken from each testing concentration at the start and at the end of each water renewal period. Controls were analysed at the start of each water renewal period. The validity criteria of the test were fulfilled.

The offspring produced by each parent animal were removed and counted daily from the appearance of the first brood. The statistical evaluation of the mean number of living juveniles showed that the offspring production for concentrations 0.44, 0.89 and 1.65 mg/L (measured) was statistically significantly different from that of the solvent control. No significant reduction of the offspring production was observed in the two lowest test concentration (0.12 and 0.22 mg/L; measured) and in the untreated control when compared to the solvent control group.

Accordingly, the 21-day NOEC related to reproduction was determined to be 0.22 mg/L (measured). The 21-day LOEC was 0.44 mg/L (measured). The 21-day EC50 was determined to be 0.58 mg/L (95 % confidence limits: 0.53 – 0.62 mg/L). The 21-day EC10 was determined to be 0.33 mg/L (95 % confidence limits: 0.28 – 0.36 mg/L).

Aborted broods were observed in some cases at the two highest concentration (0.89 and 1.65 mg/L; measured). Stumpy limbs of parent animals were noticed in the highest test concentration (1.65 mg/L; measured) at the end of the study (on 21st day). The mortality among the parent animals was recorded daily. Significant mortality was observed in the two highest concentration (0.89 and 1.65 mg/L; measured) when compared to the solvent control, therefore mortality in these concentrations were considered as a test item effect and the produced number of juveniles in replicates where parental mortality occurred were also included in the data analysis related to reproduction. In the lower concentrations (as well as in the controls) no significant parent mortality was observed (based on the statistical evaluation) and therefore the replicates of dead parents were excluded from reproductive performance analysis. At the end of the test body length measurements of the parent animals were performed and the obtained data was statistically evaluated. Statistically significant differences were observed for concentrations 0.89 and 1.65 mg/L (measured), when compared to the solvent control group.