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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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 in an earlier 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: at t=0, t=24 and t=72
Volume: 2.4 mL
Storage: samples were stored in a freezer until analysis.

At the end of the exposure period, 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 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 filtered through a 0.45 µm membrane filter (Whatman; RC55). 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. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.
- Controls: test medium without test item or other additives (blank-control)

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture.

CULTIVATION:
- Method: algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm
- Medium different from test medium: yes, M1

- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium different from test medium: no, M2

ACCLIMATION
- Acclimation period: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3 mg/L

Test temperature:

1st test: 22 - 23°C
2nd test: 21 - 23°C

pH:

1st test: 4.7 - 8.2
2nd test: 5.1 - 8.1
(see table 1 and 2 in the section 'Any other information on material and methods' for more details)

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
Mean measured test concentrations: in the first test, no detectable test item concentrations were found at a detection limit of 0.019 mg/L. Only in the second test a concentration of 1.59 mg/L was found at t=0 for a loading rate of 100 mg/L, this concentration decreased below the LOD (<0.023 mg/L) within 24 hours of exposure.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, open, fill volume: 50 mL
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 188 x 10^4 cells/mL
- No. of vessels per intermediate concentration (replicates): 3 + 1 for sampling purposes after 24 h
- No. of vessels per control and highest concentration (replicates): 6 + 1 for sampling after 24 h
- 1 or 2 replicates of each concentration without algae was used.

TEST MEDIUM / WATER PARAMETERS
- Standard test medium used: yes, M2
- Source of dilution water: Milli-RO water
- Culture medium different from test medium: yes, M1

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: continuously using TLD-lamps with a light intensity within the range of 89 to 91 µE/m2/s (1st test) and 82 to 88 µE/m2/s (2nd test).

EFFECT PARAMETERS MEASURED: growth rate and yield at 72 hours.
- Additional measurements: pH at the beginning and at the end of the test in control and all test concentrations, temperature of the medium continuously in a temperature control vessel, appearance of the cells at the end of the final test by microscopic observations.
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank and the extra replicates without algae as background for the treated solutions. Cell densities in the 2nd combined limit/range-finding test were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (July 2016)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes, in both tests
- No biological, behavioural or other abnormalities were observed.
- Effect concentrations exceeding solubility of substance in test medium: yes

Temperature values remained within acceptable limits throughout the duration of the study. The pH generally remained within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 units). However, it should be noted that the pH measured in the highest test concentration at the end of the test had decreased below 6.0 in both tests without having a significant effect on algal growth. The reason for the pH shift was concluded to be related to the high instability of the test item in water.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50: 1.0 mg/L, 95%CI: 0.97-1.0 mg/L
- EyC50: 0.43 mg/L, 95%CI: 0.42 - 0.44 mg/L
- Other: results fell within the historical range.

Reported statistics and error estimates:

Statistical analysis was performed only on the control and the highest test concentration. An effect was considered to be significant if statistical analysis of the data obtained for the highest test concentration compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test Procedure, a=0.05, one-sided, smaller).

No EC10,50-values could be calculated because the test item proved to be non-toxic (EC50 > maximum concentration tested).

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

Table1 Percentage inhibition of growth rate(1^st test)

Carbaethoxyphthalimid % SS prep. at 100 mg/L	Mean	Std. Dev.	N	% inhibition
Control	1.744	0.0291	6
1.0	1.774	0.0163	3	-1.7
10	1.761	0.0104	3	-1.0
100	1.616	0.0050	6	7.4 ^a

Table 2 Percentage inhibition of yield (1^st test)

Carbaethoxyphthalimid % SS prep. at 100 mg/L	Mean	Std. Dev.	n	% Inhibition
Control	187.0	16.95	6
1.0	203.8	9.86	3	-9.0
10	196.0	6.19	3	-4.8
100	126.4	1.90	6	32.4 ^a

^a statistically significant

Table 3 Percentage inhibition of growth rate (2^nd test)

Carbaethoxyphthalimid % SS prep. at 100 mg/L	Mean	Std. Dev.	n	% Inhibition
Control	1.587	0.0418	6
1.0	1.586	0.0251	3	0.1
10	1.604	0.0468	3	-1.1
100	1.588	0.0187	6	-0.1

Table 4 Percentage inhibition of yield (2^nd test)

Carbaethoxyphthalimid % SS prep. at 100 mg/L	Mean	Std. Dev.	n	% Inhibition
Control	116.6	14.86	6
1.0	115.7	8.96	3	0.8
10	122.9	17.88	3	-5.4
100	116.5	6.36	6	0.1

Table 5 Effect parameters

Parameter (mg/L)*	NOEC	EC10	EC20	EC50
Growth rate	100	>100	>100	>100
Yield	<100	<100	<100	>100

*100% SS (filtrate)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

1). In the control, cell density increased > 16 fold. 2). Mean CV for section-by-section specific growth rates in the control cultures < 35%. 3). CV of average specific growth rates during the whole test period in replicate control cultures < 7%.

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, no significant inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of Carbaethoxyphthalimid tested.
Both the EC50 for growth rate inhibition (72h-ERC50) and the EC50 for yield inhibition (72h-EYC50) were above the limit of solubility (=100% SS), corresponding with an initial measured concentration of 1.59 mg/L that decreased within 24 hours below the LOD (<0.023 mg/L).

Both the 72h-NOEC for growth rate inhibition and the 72h-NOEC for yield inhibition were at or above the limit of solubility (=100% SS), corresponding with an initial measured concentration of 1.59 mg/L that decreased within 24 hours below the LOD (<0.023 mg/L).

Due to the very low solubility of Carbaethoxyphthalimid in test medium, concentration levels toxic for algae could not be reached. It was concluded that Carbaethoxyphthalimid did not affect growth of the fresh water algae species Pseudokirchneriella subcapitata 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 growth rate and yield of fresh water algae ( Pseudokirchneriella subcapitata ) after 72 hours of exposure. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 201 and GLP under static conditions.

Two combined limit/range-finding test were performed. For each test, six exponentially growing algal cultures were exposed to an untreated control and the limit concentration, i.e. In addition, three replicates per group were exposed to 1.0 and 10 mg/L. The initial algal cell density was 10⁴cells/mL. The total exposure period was 72 hours. Samples were taken and analysed at the start, after 24 and 72 hours of exposure. In the first test, no detectable test item concentrations were found at a detection limit of 0.019 mg/L. Analysis of the undissolved residue on the filter proved that the test substance was used during formulation. Hence, the solubility of the test item in test medium after one day of stirring was so low that quantification proved impossible. In the second test, a concentration of 1.59 mg/L was found at t=0.This concentration decreased below the LOD (<0.023 mg/L) within 24 hours of exposure. This concentration was expected to represent the maximum soluble concentration of Carbaethoxyphthalimid in test medium after 2 hours of stirring.

No significant inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of test item tested. Both the EC₅₀ for growth rate inhibition (72h-E_RC₅₀) and the EC₅₀ for yield inhibition (72h-E_YC₅₀) were above the limit of solubility (=100% SS). This corresponds with an initially measured concentration of 1.59 mg/L that decreased within 24 hours below the LOD (<0.023 mg/L).

Both the 72h-NOEC for growth rate inhibition and the 72h-NOEC for yield inhibition were at or above the limit of solubility (=100% SS), corresponding with an initial measured concentration of 1.59 mg/L that decreased within 24 hours below the LOD (<0.023 mg/L). 

Due to the very low solubility of the test item in test medium, concentration levels toxic for algae could not be reached. It was concluded that Carbaethoxyphthalimid did not affect growth of the fresh water algae species Pseudokirchneriella subcapitata up to the maximum soluble concentration in test medium.