Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
April 2008
Reliability:
3 (not reliable)
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).

Section-by-section growth rates (i.e., 0-24 h, 24-28 h and 48-72 h) in the control cultures

In section "Comparison of Growth Rates" of the report, it is stated that the section-by-section specific growth rate (days 0-1, 1-2, and 2-3) were calculated for the control cultures and the results were examined to determine whether the growth rate remained consistent.

Daily Specific Growth Rates for the Control Cultures in theDefinitive Test

 

Daily specific growth rate

Control

0-24h

24-48h

48-72h

R1 -

0.047

0.057

0.038

R2

0.049

0.051

0.05

R3

0.045

0.063

0.049

R4

0.041

0.055

0.045

R5

0.039

0.058

0.05

R6

0.04

0.06

0.05

Mean

0.044

0.057

0.047

Daily Specific Growth Rates for the Control Cultures in thePositive Control

 

Daily specific growth rate

Control

0-24h

24-48h

48-72h

R1 -

0.062

0.047

0.058

R2

0.061

0.049

0.063

R3

0.064

0.049

0.06

R4

0.069

0.044

0.05

R5

0.064

0.052

0.057

R6

0.071

0.047

0.056

Mean

0.065

0.048

0.057

The following data show that the cell concentration of the control cultures increased by a factor of 36 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

- Mean cell density of control at 0 hours: 4.12 x 103 cells per ml

- Mean cell density of control at 72 hours: 1.46 x 105 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 16% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

 

The initial biomass and the biomass at the end of the test

- The mean cell density at initiation (0 h) was 4.12 x 10 Exp.3 cells per ml

- The mean cell density at termination in the control (72 h) was 1.46 x 10 Exp.5 cells per ml

- Cell concentrations in the control cultures increased by a factor of 36.

 

The mean coefficient of variation for section-by-section specific growth

The mean coefficient of variation for section-by-section growth rate for the control cultures was 16%.

 

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 h) was 4%.

 

Technical specifications impacting the sensitivity/reliability of the test

The test medium used was reconstituted freshwater culture medium (= AAP medium). The constitutents of the culture medium are the same as those listed in OECD TG 201 for preparation of AAP medium

NaN03

25.5 mg/l

MgCl2x 6 H20

12.164 mg/l

CaCl2x 2 H20

4.41 mg/l

MgS04x 7 H20

14.7 mg/l

K2HP04

1.044 mg/l

NaHC03

15.0 mg/l

H3B03

0.1855 mg/l

MnCl2x 4 H20

0.415 mg/l

ZnCl2

0.00327 mg/l

FeCl3x 6 H20

0.159 mg/l

CoCl2x 6 H20

0.00143 mg/l

Na2Mo04x 2 H20

0.00726 mg/l

Na2Mo04x 2 H20

0.000012 mg/l

CuCl2x 2 H20

0.03 mg/l

Na2EDTA x 2 H20

0.30 mg/l

Na2Se03x 5 H20

0.00010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.

 

Characterisation of exposure: Performance parameters of the analytical method

Details are given in the Section "Details on analytical methods" above. All necessary paparameters were provided. The limit of quantification was determined to be 0.079 mg/l.

Reporting of the methodology and results: The method used to determine the algal biomass

Evaluation of data included a description of the determination of the endpoint criteria including average specific growth rate, yield as calculation of the increase in biomass ober the exposure period and biomass integral described as the area under the curve.

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.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2021 - January 2022
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)
Version / remarks:
(adopted 2006, corrected 2011)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Storage Conditions: Store in cool place. Keep container tightly closed in a dry and well-ventilated place
Analytical monitoring:
yes
Details on sampling:
For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control.
For sampling at the end of the test, the test medium of the treatment replicates was pooled.
All samples were stored frozen (at about -20 °C) immediately after sampling until analysis.
The concentrations of the test item were determined in one of the duplicate test medium samples from the two highest nominal concnetrations of 50 and 100 mg/L. The samples from the nominal test concentrations of 6.25 to 25 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test. From the control samples, one of the duplicate samples was analyzed per sampling time.
Vehicle:
no
Details on test solutions:
- Method: The test medium of the highest nominal concentration of 100 mg/L was prepared by mixing 50.07 mg of the test item into 500 mL test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. The adjusted test medium of the highest test concentration was diluted with test water to obtain the test media of the additional test item concentrations , 50, 25, 12.5 and 6.25 mg/L.
- Controls: test water only
- Test concentration separation factor: 2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum, occasionally also listed as Raphidocelis subcapitata)
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany)
- Age of inoculum (at test initiation): An inoculum culture was set up three days before the start of the exposure.
- Method of cultivation: The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: none reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
none
Post exposure observation period:
none
Hardness:
The water hardness (calculated) of the test water was 0.15 mmol/L (= 15 mg/L as CaCO3).
Test temperature:
The water temperature during the test was maintained at 22 °C.
pH:
The pH was 7.0 in the control at test start and 7.5 at test end.
The pH of the test media was in the range of 6.7 to 7.5 during the test period for all test concentrations.
Dissolved oxygen:
Not reported
Salinity:
Freshwater used
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal concentrations of 0, 6.25, 12.5, 25, 50 and 100 mg/l were used in the main test.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Material, size, headspace, fill volume: glass. 75 mL, filled with 30 mL test solution
- Aeration: Each test flask loosely covered with a glass lid to allow adequate transfer of CO2 from the surrounding air to the test solution
- Initial cells density: 5000 cells/mL (0.921 E+04 relative fluorescene units)
- Control end cells density: 586,319 cells/mL (108 E+04 relative fluorescene units)
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): six

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (AAP Medium) prepared according to the test guideline by dissolving analytical grade salts in sterile purified water.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: sterile water used to prepare test solutions
- Adjustment of pH: pH of the highest concentration solution which was then further diluted to produce the other test concentrations was adjusted to pH 6.5 at the start of the test.
- Photoperiod: 24 h light/day
- Light intensity and quality: The light intensity at the level of the test solutions was approximately 67 μE s-1 m-2 (range: 64 to 69 μE s-1 m-2, measured at nine places in the experimental area).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter at an excitation of 440 nm and emission of 680 nm, daily

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0, 4.6, 10, 46, 100 mg/L
- Results used to determine the conditions for the definitive study: Concetration/Inhibition of Yield after 72 hours (%)/Inhibition of Average Growth after 72 hours (%): Control/0.0/0.0; 4.6/1.4/0.3; 10/6.6/1.4; 46/9.0/1.9; 100/15/3.4

CULTURING APPARATUS
-Details on culturing apparatus used: The test flasks were incubated in a temperature controlled orbital shaker (Multitron-Pro, Infors HT, Bottmingen/Switzerland) at a temperature of 22 °C.
Reference substance (positive control):
yes
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
49.064 mg/L
95% CI:
>= 33 - <= 88
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
.- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test item concentration of 100 mg/L and the algal cells in the control. The shape and size of the algal cells were not affected by the test item up to this concentration.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Relevant tables from this study are provided below. Figures are attached.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The 72-hour EC50 for growth rate in the reference test IES Study Number 20210289 (May 2021) was 1.0 mg/L and showed that the sensitivity of the test system was within the range recommended by the guideline (72-hour EC50 for the growth rate 0.92-1.5 mg/L).
Reported statistics and error estimates:
Williams multiple sequential t-test procedure

Table 1 Biomass of Algae

Nominal Test Item Concentration [mg/L]

Rep. No.

 

Biomass of Algae*

 

24 hours

48 hours

72 hours

Control

1

4.03

26.0

112

2

3.38

23.3

96.5

3

3.38

23.7

102

4

4.06

25.5

113

5

4.51

27.1

111

6

4.12

26.6

114

Mean

3.91

25.4

108

SD

0.449

1.53

7.16

6.25

1

4.27

26.2

109

2

3.63

28.9

122

3

4.48

26.0

114

Mean

4.13

27.1

115

SD

0.444

1.64

6.73

12.5

1

3.74

28.6

123

2

3.84

24.8

113

3

4.08

24.2

112

Mean

3.88

25.9

116

SD

0.176

2.42

5.94

25

1

4.30

24.9

113

2

3.87

24.1

108

3

4.21

24.5

97.6

Mean

4.13

24.5

106

SD

0.229

0.441

7.97

50

1

3.70

24.6

106

2

3.70

23.5

103

3

3.46

22.6

105

Mean

3.62

23.6

105

SD

0.139

1.01

1.37

100

1

3.02

18.7

77.2

2

3.26

22.2

84.5

3

3.38

23.4

99.3

Mean

3.22

21.4

87.0

SD

0.186

2.46

11.3

SD: Standard deviation

*: The biomass was determined by fluorescence measurement (mean of duplicate measurements per replicate) and is given as relative fluorescence units (x 104). At the start of the test, the initial cell density was 5000 algal cells/mL, corresponding to 0.921 x 104relative fluorescence units.

Table 2 Average Growth Rates (μ)

Nominal Test Item Concentration [mg/L]

Average Growth Rate μ [day-1] and Inhibition Ir[%]

0-24 h

0-48 h

0-72 h

μ [day-1]

Ir[%]

μ [day-1]

Ir[%]

μ [day-1]

Ir[%]

Control

1.441

0.0

1.657

0.0

1.588

0.0

6.25

1.496

-3.8

1.690

-1.9

1.609

-1.3

12.5

1.439

0.2

1.666

-0.5

1.611

-1.4

25

1.500

-4.1

1.641

1.0

1.582

0.4

50

1.368

5.1

1.621

2.2

1.578

0.7

100

1.251*

13.2

1.571*

5.2

1.514*

4.7

*: mean value statistically significantly lower than in the control (according to Williams t-test, one-sided smaller,α= 0.05)

Table 3 Average Growth Rates (μ) in the Control Replicates after 72 Hours

Control

Replicate

Average Growth Rate μ after 72 Hours

1

1.602

2

1.551

3

1.571

4

1.603

5

1.598

6

1.607

Mean

1.588

CV [%]

1.4

Table 4 Yield (Y)

Nominal Test Item Concentration

Yield Y (x 104) and Inhibition Iy [%]

0-24 h

0-48 h

0-72 h

[mg/L]

Y

Iy [%]

Y

Iy [%]

Y

Iy [%]

Control

2.99

0.0

24.5

0.0

107.4

0.0

6.25

3.21

-7.1

26.1

-6.9

114.1

-6.3

12.5

2.96

1.0

24.9

-1.9

114.9

-7.0

25

3.21

-7.3

23.6

3.6

105.3

2.0

50

2.70

9.8

22.7

7.4

103.8

3.4

100

2.30*

23.1

20.5*

16.2

86.1*

19.9

*: mean value statistically significantly lower than in the control (according to Williams t-test, one-sided smaller,α= 0.05)

Table 5 Average Section-by-Section Growth Rates

Nominal Test Item Concentration

[mg/L]

Section-by-Section Growth Rates [day-1] and Inhibition Ir [%]

0-24 h

24-48 h

48-72 h

μ [day-1]

Ir [%]

μ [day-1]

Ir [%]

μ [day-1]

Ir [%]

Control

1.441

0.0

1.874

0.0

1.451

0.0

6.25

1.496

-3.8

1.883

-0.5

1.448

0.2

12.5

1.439

0.2

1.893

-1.1

1.502

-3.5

25

1.500

-4.1

1.782

4.9

1.464

-0.9

50

1.368

5.1

1.874

0.0

1.491

-2.8

100

1.251

13.2

1.891

-0.9

1.401

3.5

Table 6 Section-by-Section Growth Rates in the Control Replicates

Control replicate

Section-by-Section Growth Rates [day-1] and Mean Specific Growth Rate (0-72 h)

0-24 h

24-48 h

48-72 h

Mean Specific Growth Rate 0-72 h

CV

0-72 h

 

μ

μ

μ

μ

[%]

1

1.475

1.865

1.464

1.602

14.3

2

1.299

1.933

1.420

1.551

21.7

3

1.301

1.949

1.462

1.571

21.5

4

1.484

1.839

1.486

1.603

12.8

5

1.590

1.791

1.413

1.598

11.9

6

1.498

1.865

1.459

1.607

12.9

Mean

-

1.588

16.0

 

Analytical Results

At the start of the exposure, the measured concentrations of 1,2,4,5-Benzenetetracarboxylic acid in the test media for the concentrations of 50 and 100 mg/L were 103 and 106 % of the nominal values, respectively. At the end of the exposure, the measured concentrations in the test media for the concentrations 50 and 100 mg/L were 105 and 104 % of the nominal values, respectively. See table 4 below for the result of test sample analysis.

Analytical Table 4 Results for Test Samples

Sampling Day/Age of Samples

[d/h]

Nominal Concentration of Test Item cnom

[mg/L]

Measured Concentration of Test Item

x

[mg/L]

Sample Preparation Factor

F

Determined Concentration of Test Item

c

% of Nominal

0/0

0 (Control)

50

100

n.d

51.7

53

1

1

2

< LOQ

51.7

106

n.a.

103

106

3/72

0 (Control)

50

100

n.d.

52.4

52.2

1

1

2

< LOQ

52.4

104

n.a.

105

104

Samples at test end were centrifuged for 5 minutes at 2465 g.

The tabulated values of the samples represent rounded results obtained by calculation using the exact raw data.

Validity of the test:

The test was valid since the following performance criteria (according to the OECD 201) were met.

- In the control, the biomass increased by a factor of 118 over 72 hours. (Criterion: increase by at least a factor of 16 within three days).

- The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 16 %. (Criterion: must not be higher than 35 %).

- The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.4 %. (Criterion: must not be higher than 7 %).

Validity criteria fulfilled:
yes
Conclusions:
In terms of growth rate, exposure of Pseudokirchneriella subcapitata to the test material gave an ErC50 (0 - 72 h) value of > 100 mg/l nominal. In terms of yield, exposure of Pseudokirchneriella subcapitata to the test material gave an EyC50(0 - 72 h) value of > 100 mg/l nominal.
Executive summary:

As PMDA is unstable in water, the corresponding hydrolysis product Pyromellitic acid (PMA) was tested.

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum, occasionally also listed as Raphidocelis subcapitata) was investigated in a 72-hour static test according to the OECD Guideline 201 (adopted 2006, corrected 2011). The nominal test item concentrations tested were 6.25, 12.5, 25, 50 and 100 mg/L. A control (test water without test item) was tested in parallel.

At the start of the exposure, the measured concentrations of the test item in the test media for the concentrations of 50 and 100 mg/L were 103 and 106 % of the nominal values, respectively. At the end of the exposure, the measured concentrations in the test media for the concentrations 50 and 100 mg/L were 105 and 104 % of the nominal values, respectively. Thus, the analytical results confirm the correct dosage and stability of the test item over the exposure period of 72 hours. Therefore, the endpoint values were reported on the nominal concentrations of the test item.

In terms of growth rate, exposure of Pseudokirchneriella subcapitata to the test item gave an ErC50 (0 - 72 h) value of > 100 mg/L. The Lowest Observed Effect Concentration based on inhibition of growth rate was 100 mg/L and the No Observed Effect Concentration was 50 mg/L.

In terms of yield, exposure of Pseudokirchneriella subcapitata to the test item gave an EyC50(0 - 72 h) value of > 100 mg/L. The Lowest Observed Effect Concentration based on yield was100 mg/L and the No Observed Effect Concentration was 50 mg/L.

Description of key information

Key study:

As PMDA, is unstable in water, the corresponding hydrolysis product Pyromellitic acid (PMA) was tested.

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum, occasionally also listed as Raphidocelis subcapitata) was investigated in a 72-hour static test according to the OECD Guideline 201 (adopted 2006, corrected 2011). The nominal test item concentrations tested were 6.25, 12.5, 25, 50 and 100 mg/L. A control (test water without test item) was tested in parallel.

At the start of the exposure, the measured concentrations of the test item in the test media for the concentrations of 50 and 100 mg/L were 103 and 106 % of the nominal values, respectively. At the end of the exposure, the measured concentrations in the test media for the concentrations 50 and 100 mg/L were 105 and 104 % of the nominal values, respectively. Thus, the analytical results confirm the correct dosage and stability of the test item over the exposure period of 72 hours. Therefore, the endpoint values were reported on the nominal concentrations of the test item.

In terms of growth rate, exposure of Pseudokirchneriella subcapitata to the test item gave an ErC50 (0 - 72 h) value of > 100 mg/L. The Lowest Observed Effect Concentration based on inhibition of growth rate was 100 mg/L and the No Observed Effect Concentration was 50 mg/L.

In terms of yield, exposure of Pseudokirchneriella subcapitata to the test item gave an EyC50(0 - 72 h) value of > 100 mg/L. The Lowest Observed Effect Concentration based on yield was100 mg/L and the No Observed Effect Concentration was 50 mg/L.

Supporting study:

As PMDA, is unstable in water, the correspondinghydrolysis product Pyromellitic acid (PMA) was tested.

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.

This test was assessed as invalid by ECHA, therefore a new study was conducted.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
50 mg/L

Additional information