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

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

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 210 (Fish, Early-Life Stage Toxicity 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 ± 0.008 g of test substance were weighed then dissolved directly in 10 litre DSW. Previous non-GLP studies on stability have revealed that the test substance is stable for up to 48 h in DSW. The obtained preparations were agitated mechanically for between 1 and
24 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 DSW can be obtained within one hour by mechanical agitation). Stock solutions of this batch of peroxide at 100 mg/L have been shown to be stable in DSW for periods up to 3 days.
The pH of the stock solution(s) were checked and found to be between 8.0 and 8.3 and were not adjusted.

Test solutions were prepared by further dilution of the stock solution with DSW. A geometric series of concentration was used. The ratio between two consecutive concentrations did not exceed 2.
Test vessels (aquaria) were filled using a flow-through system from the test solution containers immediately after preparation.
The pH of the test solutions were between 7.6 and 8.1 and close to the value of test water (± 0.5 units).

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

- Origin: Broodstock: Dierenspeciaalzaak Engelen, Rijnstraat 17, 6811 EW Arnhem, The Netherlands. The broodstock are maintained in Akzo Nobel
Environmental Chemistry laboratory.
Eggs, Akzo Nobel Environmental Chemistry laboratory. Fertilized fish eggs collected in the laboratory were used to start the test as soon as possible after laying. The developmental phase of the eggs at test initiation was between zygote and blastodisc cleavage stage equivalent to about 45 minutes after spawning under laboratory conditions.
- Acclimatization: no acclimatization period as the test should start as soon as possible after the eggs have been fertilized.
- Allocation to study: fertilized eggs were selected using a pipette or a hand-net, without preference, and randomly assigned to test vessels.
- Number of eggs: at least 80 fertilized eggs per concentration divided into two replicates of at least 40 eggs each.
- Feeding: Larvae were fed at the free-swimming stage (approximately 4 days after hatch) with protozoa from an infusorium containing mainly Paramecia species. The protozoa were captured and pipetted into an appropriate quantity of DSW before being bottled and included in the test set up as food for the fish. Feeding started on day 5 of the test. The first day of feeding the larvae received 3 times 10 ml, on day 6 to 14 of the test 4 times 10 ml was given every day. On day 15 to 17 the larvae received again 3 feedings per day. Ten ml of food was automatically transferred using a peristaltic pump, into the test vessels until the fish were old enough to accept brine shrimp nauplii. On day 18 of the test the juveniles in the control were old enough to eat brine shrimp nauplii, but in the test substance concentrations some animals were too small to be able to eat the nauplii at this time, therefore all aquaria received 2 to 3 droplets of brine shrimp nauplii from a Pasteur pipette and 10 ml Paramecia per day until day 32 of the test. From day 33 until the end of the test only nauplii were fed.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

36 d

Test temperature:

24.4 - 26.2°C
The actual temperature was kept constant within ±1.5°C between successive days.

pH:

7.6 - 8.1

Dissolved oxygen:

7.1 - 8.4 mg 02/L.

Nominal and measured concentrations:

Nominal: 0.75, 1.5, 3.0, 6.0 and 12 mg/L.
Measured: 0.65, 1.47, 2.62, 4.93, 9.75 mg/L
The measured concentrations were close to nominal, therefore nominal concentrations were used in the calculations of effect concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Monoblock glass aquaria with a holding capacity of 1.5 Iitres
- Type (delete if not applicable): open
- Aeration: No

TEST MEDIUM / WATER PARAMETERS
Dutch Standard Water (DSW) was used for the study. A known quantity of the demineralised water passed directly into a reservoir tank and the appropriate hardness was obtained by adding salts to water. The pH of the resulting solution was between 6.0 and 8.5 (generally in the range of 8.0 ± 0.5) and conductivity measured at the beginning of the test was 561IJs/cm, which is in the range of 550 and 650 IJs/cm given for DSW. Water hardness is measured monthly in the production tank to verify that it meets the criteria. During the test it was measured in the DSW tank on day 11 and found to be 13.1 °dH (equal to 234 mg CaCO3/L) which is within the accepted range of 140 to 250 mg CaCO3/L.
- 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) :
In all test vessels, the initial stage of embryo development at the start of study was recorded. Eggs, embryos, larvae and juveniles were checked for visible abnormalities (abnormal appearance and behaviour) and mortality each day.
Criteria for death were as follows:
- for eggs: particularly in the early stages, a marked loss of translucency and change in colouration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance,
- for embryos: absence of body movement and/or absence of heart-beat,
- for larvae and juvenile fish: immobility and/or absence of respiratory movement and/or absence of heart-beat and/or white opaque colouration of central nervous system and/or lack of reactions to mechanical stimulus.
Dead embryos, larvae and juvenile fish were removed as soon as observed.
The other criteria used were as follows:
- the time to start and end of hatch and number hatched,
- the length of all surviving fish to the nearest 0.1 mm at the end of the test,
- the individual weight of these fish (blotted wet weight), was not possible. So fish were divided into
2 groups per aquarium and weighed (to calculate the mean individual wet weight of the surviving animals per replicate) to the nearest 0.1 mg,
- the number of deformed larvae,
- the number of larvae behaving abnormally.

RANGE-FINDING STUDY
No range finding was performed, concentration range was set on the basis of the acute toxicity test with fish (Notox, 2002).

Reference substance (positive control):

not specified

Key result

Duration:

36 d

Dose descriptor:

EC10

Effect conc.:

8.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: post hatch survival

Remarks on result:

other: 95% C.I. 7.0-9.6 mg/L

Key result

Duration:

36 d

Dose descriptor:

NOEC

Effect conc.:

6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: post hatch survival

Key result

Duration:

36 d

Dose descriptor:

LOEC

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: post hatch survival

Key result

Duration:

36 d

Dose descriptor:

NOEC

Effect conc.:

6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Details on results:

First hatch was observed on day 3 of the study in the control and all test concentration. Hatching in the control was complete by day 6 and in the test concentrations by day 7. Not all fish (approximately 30) hatched at all concentrations.

In the control 23 coagulated eggs were counted in total. In all test concentrations the counted number was lower; 6 to 18 coagulated eggs. It was not always possible to make a proper count, even though the counts were made 6 days a week, the delicate nature of the biological material meant that decomposition occurs very quickly. It is possible that on the one day in the week they were not counted, some coagulated eggs were already decomposed and therefore not included in the final result. Because of this uncertainty no statistics were performed on number of living eggs per group.
Despite this it can be concluded that no concentration related mortality occurred, the control having the greates count of coagulated eggs.

The numbers of fish present after swim-up in the control and thinning were reworked from the number alive at the end of the test
and the dead fish counted during the test. This was done because it was not possible to count the living fish accurately and determine the exact number of fish after thinning.
One fish out of 50 died in the control during the test. In 0.75 mg/L 1 fish out of 53 died, in 1.5 mg/L no fish died, in 3.0 mg/L 4 fish out of 62 died, in 6.0 mg/L 2 fish out of 59 died and in 12 mg/L 17 fish out of 57 died during the test.

Weighing of the fish was carried out. The fish were blotted dry and wet weight was measured. During the weighing process it was noticed that loss of water occurred, this led to significant variations in weight and therefore these results could not be used.

Reported statistics and error estimates:

The No Observed Effect Concentration (NOEC) for survival was derived as the first concentration below the LOEC value, where survival showed no significant difference to the control values, using William's test. An EC10 was determined by maximum likelihood regression using the probit transformation.
Confidence limits were computed on the basis of Fieller's theorem. All computations on survival were performed using the TOXCALCTM version 5.0 program.

Validity criteria fulfilled:

yes

Conclusions:

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.

Pre-hatch mortality was found in all concentrations, but was not as high as in the control and was therefore not concentration related. The NOEC for this endpoint is >= 12 mg/L.

Post-hatch survival showed a concentration related effect and an EC10 of 8.6 mg/L was determined.
Using ANOVA a LOEC of 12 mg/L was calculated and the NOEC is therefore 6.0 mg/L for this endpoint.

No teratogenic malformations were noted for any larvae at any concentration.

Length data were statistically assessed using multi-comparison tests. The LOEC was found to be 12 mg/L and the NOEC is therefore 6.0 mg/L.

Based on the overall results from pre-hatch mortality, post-hatch survival and length, the Lowest Observed Effect Concentration (LOEC) was considered to be 12 mg/L and the No Observed Effect Concentration (NOEC) was determined as 6.0 mg/L.

Executive summary:

The purpose of this study was to assess the toxicity of the test substance dissolved in fresh water, on the early life stages of Danio rerio, in a 36-day flow-through test complying with the OECD Guideline No. 210, 17 July 1992. The test criterion of toxicity used was the effects on hatching, larvae mortality, morphological abnormalities and growth of Danio rerio exposed to the test substance over the test period.

The nominal concentrations used in the study were as follows: 0, 0.75, 1.5, 3.0, 6.0 and 12 mg/L

Analytical determinations of the test solutions were made on 20 occasions during the test. The concentrations were found to remain stable to within 20% of the nominals. The nominal concentrations were used to calculate the effect concentrations.

The validity criteria were respected:

• the dissolved oxygen concentration was between 60 and 100% of the air saturation value throughout the test,

• water temperature remained between 23 and 2rC over the test period and did not differ more than ±1.5°C between successive days

• The post-hatch success (until the end of the test) was greater than 70% in the control.

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.

Pre-hatch mortality was found in all concentrations, but was not as high as in the control and was therefore not concentration related. Post-hatch survival showed a concentration related effect and therefore an EC10 was determined of 8.6 mg/L. The LOEC was considered to be 12 mg/L and the NOEC therefore 6.0 mg/L. No teratogenic malformations were noted for any larvae at any concentration. Length data were statistically assessed using multi-comparison tests. The LOEC was found to be 12 mg/L and the NOEC therefore 6.0 mg/L.

Based on results from length, the Lowest Observed Effect Concentration (LOEC) was considered to be 12 mg/L and the No Observed Effect Concentration (NOEC) was determined 6.0 mg/L.

Description of key information

An EC10 was determined of 8.6 mg/L. The LOEC was considered to be 12 mg/L and the NOEC therefore 6.0 mg/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

8.6 mg/L