Ethyl 1,3-dihydro-1,3-dioxo-2H-isoindole-2-carboxylate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 May 2016 - 19 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23.

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 1,3-dihydro-1,3-dioxo-2H-isoindole-2-carboxylate

EC Number:

245-048-5

EC Name:

Ethyl 1,3-dihydro-1,3-dioxo-2H-isoindole-2-carboxylate

Cas Number:

22509-74-6

Molecular formula:

C11H9NO4

IUPAC Name:

ethyl 1,3-dioxo-2,3-dihydro-1H-isoindole-2-carboxylate

Test material form:

solid

Details on test material:

- Appearance: white solid
- Storage conditions of test material: in refrigerator (2-8°C)

Specific details on test material used for the study:

- Solubility in water: 0.432 g/L at 20°C by the flask method, determined at stirring times of 1 and 2 hours due to degradation of test item in water (determined at previous study)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test concentrations and the control according to the schedule below.
Frequency: 1st test: at t=0 and t=48
2nd test: at the start and end of both 24h renewal intervals.
Volume: 2.4 mL from the approximate centre of the test vessels
Storage: samples were stored in a freezer until analysis.

At the end of the exposure period (1st test) or at the end of the renewal periods (2nd test), the replicates were pooled at each concentration before sampling.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: Two combined limit/range-finding tests were performed. Preparation of test solutions for both tests started with a loading rate of 100 mg/L. In the 1st test, a one-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. Stirring time in the 2nd test was reduced to 2 hours to assess whether there was a difference in effects as information became available after the 1st test that the test item degrades very quickly in water. The resulting dispersions were consequently filtered through a 0.45 µm membrane filter (Whatman; RC55) to remove any non-dissolved material. The obtained Saturated Solutions (SS) were used as the highest test concentrations. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.
- Controls: test medium without test item or other additives (blank-control)

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus, 1820
- Source: in-house laboratory culture with a known history. At least third generation, obtained by acyclical parthenogenesis under specified breeding conditions. Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Age at study initiation: < 24 hours, from parental daphnids of more than two weeks old.
- Feeding during test: no

ACCLIMATION
- Acclimation period: no

BREEDING:
- Method: each batch was started with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel. After 7 days of cultivation half of the medium was renewed twice a week.
- Medium other than test medium: yes, M7
- Feeding: daily, a suspension of fresh water algae.

Study design

Test type:

other: 1st test: static, 2nd test: semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Test temperature:

1st test: 20°C
2nd test: 19 - 21°C

pH:

1st test: 6.7 - 8.1 throughout the test
2nd test: 6.6 - 8.1 throughout the test

Dissolved oxygen:

1st test: 8.7 - 9.4 mg O2/L throughout the test
2nd test: 8.8 - 9.2 mg O2/L throughout the test

Nominal and measured concentrations:

Two combined limit/range-finding tests were performed.
Nominal test concentrations: 1.0, 10 and 100 % of a SS prepared at 100 mg/L
Measured concentrations first test: at a loading rate of 100 mg/L at t=0: 0.028 mg/L (calculated by extrapolation of calibration curve). Other concentrations were below detection limit of 0.019 mg/L. The average exposure concentration was calculated to be 0.016 mg/L.
Measured concentrations second test: at a loading rate of 100 mg/L at t=0: 2.37 mg/L and at t=24 (fresh solution): 2.59 mg/L. Other concentrations were below detection limit of 0.023 mg/L. The average exposure concentration was calculated to be 0.17 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass, open, fill volume: 80 mL
- Aeration: no
- Renewal rate of test solution in the second test: 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Standard medium used: yes, adjusted ISO medium
- Source/preparation of dilution water: RO-water
- Culture medium different from test medium: yes, M7

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours, daily

EFFECT PARAMETERS MEASURED: mobility at 24 and 48 hours.
- Additional measurements: pH and dissolved oxygen in the 1st test at the beginning and at the end of the test, for the highest concentration and the control. In the 2nd test all fesh and old solutions from at least the control and the highest concentration. Temperature of the medium: continuously in a temperature control vessel beginning at the start of the test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (July 2016)

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: Based on the average measured exposure concentration of the 2nd test.

Details on results:

- Effect parameters were based on the average measured exposure concentration of the 2nd test and the loading rate initially introduced. This was calculated as the average of the geometric mean of the concentrations of test substance measured in the samples taken at t=0, at t=24 in the old and in the fresh medium and at t=48. In case concentrations measured were below the limit of detection, the final exposure concentrations were taken as a factor of 2 below the applicable limit.
- No biological, behavioural or other abnormalities were observed.
- Effect concentrations exceeding solubility of substance in test medium: yes

Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 24h-EC50: 0.55 mg/L, 95%CI: 0.50-0.62 mg/L
- 48h-EC50: 0.39 mg/L, 95%CI: 0.33-0.44 mg/L
- Other: results fell within the historical range.

Any other information on results incl. tables

Table 1 Concentrations of the test item in test medium – combined limit/range-finding test 1

Time of sampling^1 [hours]	Percentage of SS^2 [%]	Analysed concentration [mg/L]
0	0	n.d.
	100	0.028^3
48	0	n.d.
	100	n.d.

¹Samples were stored in the freezer (= -15°C) until the day of analysis.

²Saturated solution prepared at a loading rate of 100 mg/L.

³Estimated value, calculated by extrapolation of the calibration curve.

n.d.Not detected. The limit of detection of the method was determined to be 0.019 mg/L taking a dilution factor of 1.67 into account.

n.a.Not applicable.

Table 2 Concentrations of the test item in test medium – combined limit/range-finding test 2

Time of sampling^1 [hours]	Percentage of SS^2 [%]	Analysed concentration [mg/L]
0	0	n.d.
(fresh)	100	2.37
1	0	n.d.
(old)	100	n.d.
1	0	n.d.
(fresh)	100	2.59
2	0	n.d.
(old)	100	n.d.

¹Samples were stored in the freezer (= -15°C) until the day of analysis.

²Saturated solution prepared at a loading rate of 100 mg/L.

n.d.Not detected. The limit of detection of the method was determined to be 0.023 mg/L taking a dilution factor of 1.67 into account.

n.a.Not applicable.

Table 3 Number of introduced daphnids and incidence of immobility(1^sttest)

Time (h)	Replicate	Carbaethoxyphthalimid % SS prep. at 100 mg/L
		Control	1.0	10	100 (0.016 mg/L)
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total introduced	20	10	10	20
24	A	0	0	0	0
	B	0	0	0	0
	C	0			0
	D	0			0
	Total immobilised	0	0	0	0
	Effect %	0	0	0	0
48	A	0	0	0	0
	B	0	0	0	2
	C	0			1
	D	0			0
	Total immobilised	0	0	0	3
	Effect %	0	0	0	15

Table3          
Number of introduced daphnids and incidence of immobility (2^ndtest)

Time (h)	Replicate	Carbaethoxyphthalimid % SS prep. at 100 mg/L
		Control	1.0	10	100 (0.17 mg/L)
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total introduced	20	10	10	20
24	A	0	0	0 (2)	0
	B	0	0	0	0
	C	0			0
	D	0			0
	Total immobilised	0	0	0	0
	Effect %	0	0	0	0
48	A	0	0	0	0
	B	0	0	0	0
	C	0			0
	D	0			0
	Total immobilised	0	0	0	0
	Effect %	0	0	0	0

( ) between brackets: number of daphnia observed trapped at the surface of the test solution. These organisms were reimmersed into the respective solution before recording of mobility.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

1). In the controls, no daphnids became immobilised or showed other signs of disease or stress. 2). The oxygen concentration at the end of both tests was = 3 mg/L in control and test vessels.

Conclusions:

Under the conditions of the present study, the 48h-EC50 was beyond the range tested, i.e. exceeded an average measured concentration of 0.17 mg/L. This concentrationwas the maximum soluble concentration in a filtrate prepared at a loading rate of 100 mg/L over the test period. Due to the very low solubility of test substance in test medium, concentration levels inducing 50% or more effect on mobility of Daphnia magna could not be reached. It was concluded that the test substance did not affect mobility of Daphnia magna up to the maximum soluble concentration in test medium.

Executive summary:

This study was performed to assess the effect of the test substance on the mobility of Daphnia magna after 48 hours. The study was conducted in accordance with OECD Guidline for Testing of Chemicals No. 202 and performed under GLP.

 

Two combined limit/range-finding tests were performed. In the first test, the test solution was stirred for one day to ensure maximum dissolution of the test item in test medium. After the first test, information became available that the test item degrades very quickly in water. Therefore, the test solution was stirred for 2 hours in a second test to assess whether there was a difference in effects.

Loading rates of 1.0, 10 and 100 mg/L were used for both tests. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control andthe limit concentration, i.e. the undiluted SS in both tests. In addition, ten daphnids per group (5 per replicate, duplicate) were exposed to1.0 and 10% SS in the combined range-finding test. The total exposure period was 48 hours. The 1st test was performed without renewal of test solutions (static), while test solutions were renewed after 24 hours in the 2nd test (semi-static). Samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the 1st test. In the 2nd test, samples were taken from the freshly prepared and from the 24-hour old solutions. The incidence of immobilisation was recorded for each test at 24 hours and at 48 hours. Analysis of the sample taken from the SS at the start of the 1st test showed a measured concentration of 0.028 mg/L. During the exposure period the measured concentration decreased below the Limit Of Detection (LOD), i.e. below 0.019 mg/L. The average exposure concentration was calculated to be 0.016 mg/L.

Analysis of the samples taken from the freshly prepared SS at the start and after 24 hours during the 2nd test showed measured concentrations of 2.37 and 2.59 mg/L, respectively. These concentrations were 100 times higher than those measured in the 1st test and expected to represent the maximum soluble concentration in test medium after 2 hours of stirring. The measured concentrations decreased below the LOD, i.e. below 0.023 mg/L, during both 24-hour renewal periods. The average exposure concentration was calculated to be 0.17 mg/L.

 

The final results were based on the average exposure concentration determined in the 2nd test. EC50 for mobility of Daphnia magna after 48 hours of exposure to the test substance exceeded the average measured concentration of 0.17 mg/L, being the maximum soluble concentration in a filtrate prepared at a loading rate of 100 mg/L over the test period.

Due to the very low solubility of Carbaethoxyphthalimid in test medium, concentration levels inducing 50% or more effect on mobility of Daphnia magna could not be reached. It was concluded that Carbaethoxyphthalimid did not affect mobility of Daphnia magna up to the maximum soluble concentration in test medium.