dimethyl 1,2-benzenedicarboxylate;reaction mass of: 1,2-dimethylpropylidene dihydroperoxide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

As the test substance is known to be unstable under the conditions of the study, samples were taken three times per week and pooled between replicates. Samples were filtered over a Pall 0.45 pm GHP Acrodisc filter, transferred into 10 ml HPLC vials and analyzed immediately. When considered
necessary, further samples were taken within 24 hours, and analyzed immediately.

Vehicle:

no

Details on test solutions:

To prepare the stock solutions, 1.00 ± 0.003 g of the test substance was weighed out, then dissolved directly into 10 litres (determined using a measuring cylinder) of M4 medium in 10 litre Duran glass bottles and mechanically agitated using a magnetic stirrer. Previous non-GLP studies on stability have revealed that the test substance is stable for up to 72 h in M4. The obtained preparations was agitated mechanically for between 4 and 47 hours in an attempt to completely dissolve the test substance (previous non-GLP studies have shown that an aqueous solution of 100 mg/L of test substance in M4 can be obtained within one hour by mechanical agitation).
Test substance stocks were made as required, on the day they needed replacing (accept for 2 occasions where it was made earlier).The pH of each stock solution was checked and found to be between 8.1 and 8.5, therefore the pH was not adjusted.
A fresh stock solution was prepared for each solution change.

Test solutions were prepared by further dilution of the stock solution with M4 medium under flow-through conditions. The stock was pumped at a known rate into the dilution water and allowed to mix directly in the inlet pipe shortly before reaching the test aquaria. This minimised contact of the test substance with the algae thereby optimising stability.
Test vessels were filled directly by a flow-through system and analysed 3 times a week. Measured concentrations were used as feedback to immediately ajust the pumps of the flow-through system to maintain the concentrations within 80 and 120% of the nominal concentrations. The pH of the test solutions was between 7.0 and 7.8 throughout the test, therefore no adjustments were made.
One control containing only test medium was included in the test.
The test aquaria were replaced after 8 days and then whenever considered necessary by the technician, thereafter (based on visual observation of algal debris on the floor of the aquarium).

Test organisms (species):

Daphnia magna

Details on test organisms:

The test animals were taken from a Daphnia magna clone 5 stock, (origin: Notox b.v., Hambakenwetering 7, P.O. Box 3476, 5203 DL s'Hertogenbosch, The Netherland~). 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).

Culture animals were fed a diet, in the form of the algal strain ChIarella vulgaris. This strain is cultured in the ECRA Environmental Chemistry laboratory and the total organic carbon content to cell count ratio has previously been determined.
During the study the daphnids were continuously supplied with algae such that the cell count per mL was equivalent to the daily feeding rate recommended in the semi-static test of 0.1 mg of carbon per daphnid per day. This rate was maintained throughout the test for the control and the test solutions containing test substance.
The algae concentrations (cells/mL) in the controls and the test solutions were checked at least once per week. The concentration of cells provided to the daphnids ranged from 112,500 to 225,000 cells/ml while the target concentration was 200,000 cells/ml. This range was considered acceptable for
the continued health of the daphnid population without leading to problems of over-feeding or significant loss of the test substance during the study.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

Conductivity: 504-700 µS/cm
Hardness: 12.3 °dH in the control; 13.2 °dH in 18 mg/L

Test temperature:

18.4-19.5 °C

pH:

7.0-7.8

Dissolved oxygen:

6.3-8.9 mg O2/L

Nominal and measured concentrations:

Nominal: 1.1, 2.25, 4.5, 9.0, 18.0 mg/L
Measured: 1.1, 2.2, 4.3, 8.7 and 18.4 mg/L

The measured concentration were close to the nominals, therefore nominal concentrations were used to calculate the effect concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 750 ml (nominal) glass aquaria were used, pierced at approximately 450 ml level with an overflow running to drain. The aquaria were filled by a flow-through system containing test solution which passed through glass tubing on the opposite side to the over-flow. The tubing used in the pumps was Viton tubing previously determined to be the best tubing for maintaining substance stability for this organic peroxide. All remaining tUbing was made of neoprene and glass in the test concentrations and silicone in the control. Joints were minimised where possible.
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): Test solutions were constantly renewed using a peristaltic pump system.
The test system was allowed to run prior to addition of the daphnids for 7 days until deemed to be stable (further to analytical measurements).
- Renewal rate of test solution (frequency/flow rate): replacement rate of at least 10 volumes of the test vessels per day.
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

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

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours of light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Animals were checked for immobilisation of parent daphnids on at least six occasions per week of the test. From the day of the first brood, observations of broods (aborted, living and dead progeny) were also made in each container at each concentration. The day of brood release and the number of living and dead neonates per brood or abortions were noted. Any other abnormal observations were also recorded.
At the end of the test, the length of all surviving parent animals was measured to the nearest 0.1 unit using a binocular microscope. Following measurement of body length, parent animals were weighed (dry weight) by placing in an oven at 105°C overnight, individually, if possible, or per group (to calculate the mean individual weight per surviving parent animals per concentration).

RANGE-FINDING STUDY
No range finding was performed, the test concentration range was based on the acute toxicity study with Daphnia magna (Notox, 2002)

Reference substance (positive control):

not specified

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

9.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: parent mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: parental length

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: parental weight

Details on results:

The following daphnids died in each of the following concentrations: 1, 2 and 3 in the control (on day 9, 16 and 18, respectively); 1 on five occasions in 1.1 mg/L (on day 8, 9, 11, 15 and 17, respectively); 1 on five occasions and then 2 in 2.25 mg/L (on day 9, 10, 11, 14, 16 and 17, respectively); 1 on three occasions and then 2 in 4.5 mg/L (on day 9, 14, 16 and 17, respectively); 1 in 9.0 mg/L (day 15); and 1, 2, 1, 5, 1, 1 and 1 in 18.0 mg/L (on day 3, 8, 10, 14, 15, 17 and 18, respectively). These mortalities were considered to be concentration related.

The number of juveniles per replicate in each concentration is shown in table 2 (see attached document). The validity criteria for the coefficient of variation (less than 25% in the control based on the. number of living neonates for each replicate at the end of the test) was achieved.

Reported statistics and error estimates:

The reproduction data was tested for normality using Chi-square test, Bartlett's tests for homogeneity of variance was used. Analysis of variance was
performed on the number of living neonates per replicate using the Bonferroni t-test and verified with a second multiple comparison method, the Dunnett's test. All computations were performed using Toxstat version 3.0.

An EC10 was determined by maximum likelihood regression using the probit transformation. Confidence limits could not be computed. All computations on survival were performed using the TOXCALC TM version 5.0 program.
Because of too high variation in the number of surviving animals between replicates and also between concentrations, the data were transformed. It was assumed that in the control no mortality occurred and all data were adjusted accordingly. This means that the number of animals at the start of the test were set to 17 for all concentrations and when the number of daphnids at the end of the test was higher than 17, this was also set to 17, which means no mortality. This transformation was used as a surrogate for the Taw data making calculation of an EC10 possible. As the mortality data are clearly concentration related for the highest test concentration, this was considered the most appropriate statistical method to evaluate this endpoint.

Validity criteria fulfilled:

yes

Conclusions:

The test data for neonate production were found to be normally distributed and homogeneous. Using Dunnett's and Bonferroni-t tests, no significant differences were found. The No Observed Effect Concentration (NOEC) based on reproductive output, weight of adult daphnids and on parent body length was found to be >= 18.0 mg/L.

The EC50 for adult mortality and for reproduction could not be determined due to insufficient mortality in any of the test concentrations.
However, the EC10 value for parental mortality was found to be 9.7 mg/L. The final result for this study is therefore based on the EC10 for parental mortality of 9.7 mg/L.

Executive summary:

The purpose of this study was to assess the toxicity of MIPKP in DMP dissolved in fresh water, on the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a 21-day flow-through test complying with the OECD Guideline No. 211, 21 st September 1998 and EU guideline C.20 from Annex V of Directive 67/548/EEC.

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

The nominal concentrations used in the study were as follows: 0, 1.1, 2.25, 4.5, 9 and 18 mg/L

Analytical determinations of the test solutions were performed on 12 occasions during the test. The concentrations were found to remain stable to within 20% of the nominaIs over the test period. The nominal concentrations were used to calculate the effect concentrations.

The validity criteria were respected:

- Mortality was <20% in the control group over the test period.

- The average number of juveniles per replicate in the control was 1916 after 21 days equivalent to at least 95.8 neonates per daphnid. Due to the test design, the actual neonate production of individual parent animals could not be ascertained.

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.

The test data for neonate production were found to be normally distributed and homogeneous. Using Dunnett's and Bonferroni-t tests, no significant differences were found. The No Observed Effect Concentration (NOEC) based on reproductive output, weight of adult daphnids and on parent body length was found to be >= 18.0 mg/L.

The EC50 for adult mortality and for reproduction could not be determined due to insufficient mortality in any of the test concentrations.

However, the EC10 value for parental mortality was found to be 9.7 mg/L. The final result for this study is therefore based on the EC10 for parental mortality of 9.7 mg/L.

Description of key information

The EC10 value for parental mortality was found to be 9.7 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

9.7 mg/L