Benzene-1,2:4,5-tetracarboxylic dianhydride

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

test item is unstable in water, therefore the hydrolysis product PMA was tested

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Description: white powder
- Batch number: 8M30N
- Storage conditions: room temperature in the dark upto 04 April 2008, thereafter at approximately 4°C, over silica gel, in the dark

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours.Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 and
72 hours and stored at approximately -20°C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

For the purpose of the definitive test, the test material was dissolved directly in culture medium. An amount of test material (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 2 minutes and the volume adjusted to 500 ml to give a 200 mg/l stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 100, 50, 25 and 12.5 mg/. . An aliquot (250 ml) of each of the stock solutions was separately mixed with algal suspension (250 ml) to give the required test concentrations of 6.25, 12.5, 25, 50 and 100 mg/l.The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test was carried out using Desmodesmus subspicatus strain CCAP 276/20. Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mi. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100- 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 104 - 105 cells/mi.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

reconstituted culture medium; hardness not mentioned

Test temperature:

temperature was maintained at 24 ± 1°C throughout the test.

pH:

test item samples: 2.8 - 7.4
control samples: 7.2 - 7.7

Dissolved oxygen:

not measured

Salinity:

freshwater used

Nominal and measured concentrations:

The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.0010, 0.010, 0.10, 1.0, 10 and 100 mg/l for a period of 72 hours. For the main study, Desmodesmus subspicatus was exposed to an aqueous solution of the test material at concentrations of 6.25, 12.5, 25, 50 and 100 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 91% to 111% of nominal and so the results are based on nominal test concentrations only.

Details on test conditions:

The test concentrations to be used in the definitive test were determined by a preliminary rangefinding test. The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.0010, 0.010, 0.10, 1.0, 10 and 100 mg/l for a period of 72 hours. The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks, each containing 100 ml of test preparation were used for each control and test concentration. The test material was dissolved directly in culture medium.
An amount of test material (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 2 minutes and the volume adjusted to 500 ml to give a 200 mg/l stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 20, 2.0, 0.20, 0.020 and 0.0020 mg/1. An aliquot (100 ml) of each of the stock solutions was separately mixed with algal suspension (100 ml) to give the required test concentrations of 0.0010, 0.010, 0.10, 1.0, 10 and 100 mg/l. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. The control group was maintained under identical conditions but not exposed to the test material. At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380- 730 nm) and constantly shaken at approximately 150 rpm for 72 hours. After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/l.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

In terms of growth rate, exposure of Desmodesmus subspicatus to the test material gave n ErC50 (0 - 72 h) value of 8.1 mg/l. The Lowest Observed Effect Concentration based on inhibition of growth rate was 12.5 mg/l and the No Observed Effect Concentration was 6.25 mg/l. In terms of yield, exposure of Desmodesmus subspicatus to the test material gave an EyC50 (0 - 72 h) value of 7.9 mg/l. The Lowest Observed Effect Concentration based on yield was 12.5 mg/l and the No Observed Effect Concentration was 6.25 mg/l. In terms of biomass integral (area under growth curve), exposure of Desmodesmus subspicatus to the test material gave an EbC50 (0 - 72 h) value of 8.8 mg/l. The Lowest Observed Effect Concentration based on inhibition of biomass integral was 12.5 mg/l and the No Observed Effect Concentration was 6.25 mg/l.

Results with reference substance (positive control):

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatus was exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/1 (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24± 1°C.
Exposure of Desmodesmus subspicatus to the reference material, potassium dichromate, gave an ErC50 (0 - 72 h) of 0.64 mg/l, an EyC50 (0 - 72 h) of 0.36 mg/l and an EbC50 (0 - 72 h) of 0.32 mg/l. The Lowest Observed Effect Concentration based on inhibition of growth rate, yield and biomass integral was 0.25 mg/l and the No Observed Effect Concentration was 0.125 mg/l.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the
SAS computer software package (SAS 1999- 2001).

Validity criteria fulfilled:

yes

Conclusions:

In terms of growth rate, exposure of Desmodesmus subspicatus to the test material gave n ErC50 (0 - 72 h) value of 8.1 mg/l. The Lowest Observed Effect Concentration based on inhibition of growth rate was 12.5 mg/l and the No Observed Effect Concentration was 6.25 mg/l. In terms of yield, exposure of Desmodesmus subspicatus to the test material gave an EyC50 (0 - 72 h) value of 7.9 mg/l. The Lowest Observed Effect Concentration based on yield was 12.5 mg/l and the No Observed Effect Concentration was 6.25 mg/l. In terms of biomass integral (area under growth curve), exposure of Desmodesmus subspicatus to the test material gave an EbC50 (0 - 72 h) value of 8.8 mg/l. The Lowest Observed Effect Concentration based on inhibition of biomass integral was 12.5 mg/l and the No Observed Effect Concentration was 6.25 mg/l.

Executive summary:

As the substance to be registered, PMDA, is unstable in water, the corresponding hydrolysis product Pyromellitic acid (PMA) was tested. The study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test". Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to an aqueous solution of the test material at concentrations of 6.25, 12.5, 25, 50 and 100 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

In terms of growth rate, exposure of Desmodesmus subspicatus to the test material gave an ErC50 (0 - 72 h) value of 8.1 mg/l. The Lowest Observed Effect Concentration based on inhibition of growth rate was 12.5 mg/l and the No Observed Effect Concentration was 6.25 mg/l.

In terms of yield, exposure of Desmodesmus subspicatus to the test material gave an EyC50(0 - 72 h) value of 7.9 mg/l. The Lowest Observed Effect Concentration based on yield was12.5 mg/l and the No Observed Effect Concentration was 6.25 mg/l.

In terms of biomass integral (area under growth curve), exposure of Desmodesmus subspicatus to the test material gave an EbC50 (0 - 72 h) value of 8.8 mg/l The Lowest Observed Effect Concentration based on inhibition of biomass integral was 12.5 mg/l and the No Observed Effect Concentration was 6.25 mg/l. Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 91% to 111% of nominal and so the results are based on nominal test concentrations only.

Description of key information

As the substance to be registered, PMDA, is unstable in water (t1/2 approx. 20 min), the corresponding hydrolysis product Pyromellitic acid (PMA) was tested. The study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test".

In terms of growth rate, exposure of Desmodesmus subspicatus to the test material gave an ErC50 (0 - 72 h) value of 8.1 mg/l. The Lowest Observed Effect Concentration based on inhibition of growth rate was 12.5 mg/l and the No Observed Effect Concentration was 6.25 mg/l.

In terms of yield, exposure of Desmodesmus subspicatus to the test material gave an EyC50(0 - 72 h) value of 7.9 mg/l. The Lowest Observed Effect Concentration based on yield was12.5 mg/l and the No Observed Effect Concentration was 6.25 mg/l.

In terms of biomass integral (area under growth curve), exposure of Desmodesmus subspicatus to the test material gave an EbC50 (0 - 72 h) value of 8.8 mg/l The Lowest Observed Effect Concentration based on inhibition of biomass integral was 12.5 mg/l and the No Observed Effect Concentration was 6.25 mg/l.

Key value for chemical safety assessment

EC50 for freshwater algae:

7.9 mg/L

EC10 or NOEC for freshwater algae:

6.25 mg/L