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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-04-14 to 2010-04-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The substance caused a decrease in pH of the test solution at the highest test concentrations. The test was repeated with the two highest test concentration (pH was adjusted).
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
* Batch manufactured and supplied by DSM fine Chemicals Austria
* Batch No.: LPMIS3A1015
- Purity: 99.7 %
- Expiration date of the lot/batch: Oct. 2013
Analytical monitoring:
yes
Details on sampling:
Test solutions of all five concentrations were analysed along with the negative control.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg/ml was prepared by mixing 250 mg with 25 ml test medium. The test solutions were prepared separately by dilution of the stock solution. Stock solution was freshly prepared and used within 15 minutes of preparation.
- Eluate: N/A
- Differential loading: N/A
- Controls: The test medium itself was used as the negative control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): N/A
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): Norwegian Institute for Water Research, Oslo, Norway
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.6–23.4 °C
pH:
Initial test: 4.64–8.31
Repeat test: 7.72–8.36
Nominal and measured concentrations:
Nominal test concentrations: 2, 5, 12.5, 31.25 and 78.13 mg/l
Mean measured concentrations: 2.04, 5.13, 12.78, 31.76, 79.72 mg/l
Repeated test: 31.25 and 78.13 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: conical flasks (250 ml)
- Fill volume: 150 ml
- Initial cells density: 10,000 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the test medium was prepared according to OECD guideline 201

OTHER TEST CONDITIONS
- Adjustment of pH: yes at 31.25 and 78.13 mg/l
- Photoperiod: continous illumination
- Light intensity and quality: 7240–7390 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell growth (haemocytometer) after 24, 48 and 72 hours of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
11.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
22.2 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.:
74.35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The substance caused a decrease of the pH in the test medium. Differences in the algal growth between the initial test and the repeated test were ascribed to the low pH at test concentrations of 31.25 and 78.13 mg/l in the initial test.
Results with reference substance (positive control):
- Reference substance: potassium dichromate
- Other: 36.59 % and 34.12 % growth rate inhibition in definitive and repeat definitive test
Reported statistics and error estimates:
The EC10, EC20 and EC50 were calculated for the definitive test.
The inhibition of growth was determined by calculating the ErC and EyC (the index “r” refers to growth rate and the index “y” refers to yield) by Probit analysis of Finney (1971) using in-house developed and validated computer programme.
The equation of growth rate and yield was Y = a + bX, where ‘X’ is the log concentration and ‘Y' is the probit value with ‘a’ and ‘b’ as constants.
Validity criteria fulfilled:
yes
Conclusions:
In a study according to OECD 201, the 72-h EC50 for maleic acid in the freshwater algae P. subcapitata was determined at 74.35 mg/L.
Executive summary:

In a 72-h toxicity study, the freshwater alga Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum) was exposed to maleic acid at nominal concentrations of 2, 5, 12.5, 31.25 and 78.13 mg/l under static conditions in accordance with OECD guideline 201. Nominal concentrations were confirmed by analytical measurements. The 72-h EC10 and EC50 values based on growth rate were 11.80 and 74.35 mg/L, respectively. No abnormalities were noted.

This toxicity study is classified as acceptable and satisfies the guideline requirement for OECD 201 toxicity study.

Results synopsis

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

Test type: Static

72 h EC10 (growth rate): 11.80 mg/L

72 h EC20 (growth rate): 22.20 mg/L

72 h EC50 (growth rate): 74.35 mg/L

72 h EC10 (yield): 4.15 mg/L

72 h EC20 (yield): 6.75 mg/L

72 h EC50 (yield): 17.17 mg/L

The information on maleic acid is used in a read-across approach in the assessment of the target substance maleic anhydride.

Upon contact with water, maleic anhydride hydrolyses rapidly (DT50 = 0.3 min), forming maleic acid. Therefore, read-across from maleic acid to maleic anhydride is justified.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Upon contact with water, maleic anhydride hydrolyses rapidly (DT50 = 0.3 min), forming maleic acid (Bunton et al., 1963). This constitutes almost instantaneous abiotic degradation, particularly in comparison to the residence/transport times in the wastewater stream. Therefore, both sewage treatment plants and subsequently surface waters will be exposed to maleic acid (if at all) instead of maleic anhydride. Accordingly, read-across of environmental effects from maleic acid to maleic anhydride is justified.
Reason / purpose for cross-reference:
read-across source
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
11.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
22.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
74.35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The substance caused a decrease of the pH in the test medium. Differences in the algal growth between the initial test and the repeated test were ascribed to the low pH at test concentrations of 31.25 and 78.13 mg/l in the initial test.
Results with reference substance (positive control):
- Reference substance: potassium dichromate
- Other: 36.59 % and 34.12 % growth rate inhibition in definitive and repeat definitive test
Reported statistics and error estimates:
The EC10, EC20 and EC50 were calculated for the definitive test.
The inhibition of growth was determined by calculating the ErC and EyC (the index “r” refers to growth rate and the index “y” refers to yield) by Probit analysis of Finney (1971) using in-house developed and validated computer programme.
The equation of growth rate and yield was Y = a + bX, where ‘X’ is the log concentration and ‘Y' is the probit value with ‘a’ and ‘b’ as constants.
Validity criteria fulfilled:
yes
Conclusions:
In a study according to OECD 201, the 72-h EC50 for maleic acid in the freshwater algae P. subcapitata was determined at 74.35 mg maleic acid/L, corresponding to 65.78 mg maleic anhydride/L.
Executive summary:

In a 72-h toxicity study, the freshwater alga Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum) was exposed to maleic acid at nominal concentrations of 2, 5, 12.5, 31.25 and 78.13 mg/l under static conditions in accordance with OECD guideline 201. Nominal concentrations were confirmed by analytical measurements. The 72-h EC10 and EC50 values based on growth rate were 11.80 and 74.35 mg/L, respectively. No abnormalities were noted.

This toxicity study is classified as acceptable and satisfies the guideline requirement for OECD 201 toxicity study.

Results synopsis

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

Test type: Static

72 h EC10 (growth rate): 11.80 mg/L

72 h EC20 (growth rate): 22.20 mg/L

72 h EC50 (growth rate): 74.35 mg/L

72 h EC10 (yield): 4.15 mg/L

72 h EC20 (yield): 6.75 mg/L

72 h EC50 (yield): 17.17 mg/L

The information on maleic acid is used in a read-across approach in the assessment of the target substance maleic anhydride.

Upon contact with water, maleic anhydride hydrolyses rapidly (DT50 = 0.3 min), forming maleic acid. Therefore, read-across from maleic acid to maleic anhydride is justified.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Information on positive control is not reported. Analytical investigations at 0 and 72 h only -> the geometric average concentration could not be determined.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Because the test conc. were analyzed for maleic acid only at 0 and 72 hours, the geometric average concentration could not be determined. Temperature of 26°C was higher than protocol (24°C) but this was determined to not affect the outcome of the test.
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
- Strain: In-house culture from Carolina Biological Supply Company
- Method of cultivation: Cultured in Algal Assay Procedure (AAP) medium at 24 ± 4 °C under continuous illumination of 3200 to 5400 lux (Duro-Test® Vita-Lite® fluorescent bulbs), with shaking rate of 100 + 10 rpm. Inoculum culture was initiated 4 days prior to testing.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
26 ± 1 °C
pH:
Initial: 7.1 to 7.5 (control mean 7.5)
72-hours: 8.5 to 8.8 (control mean 8.7)
Conductivity:
Conductivity ranged from 80 µohms/cm (control) to 390 µohms/cm at initiation and from 80 µohms/cm (control) to 370 µohms/cm at 72 hours.
Nominal and measured concentrations:
Nominal concentrations (mg/L): 0, 9.4, 19, 38, 75, 150
Measured concentrations (mg/L)(At initiation): 0, 9.6, 19, 36, 73, 150
Analytical measurements at 72 hours showed non-detectable concentrations in the 0 to 38 mg/L treatments. Concentration in the highest treatment was 110 mg/L at 72 hours, so the arithmetic average concentration was 130 mg/L [150 mg/L and 100 mg/L at 0 and 72 hours, respectively].
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml glass beakers
- Fill volume: 100 ml
- Initial cells density: 10,000 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: AAP media.
- Conductivity: 80 μohms/cm (control) - 390 μohms/cm at start and 80 μohms/cm (control) - 370 μohms/cm at termination
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
130 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 150 mg/L
Nominal / measured:
meas. (initial)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
150 mg/L
Nominal / measured:
meas. (initial)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 150 mg/L
Nominal / measured:
meas. (initial)
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
150 mg/L
Nominal / measured:
meas. (initial)
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): Control response was satisfactory.
- Observation of abnormalities (for algal test): Microscopic observations showed no abnormal cells in any treatment.
Reported statistics and error estimates:
Exposure calculated as arithmetic mean of measurements
Statistical methods:
Growth rate calculated as: (ln Xt – ln X0)/(tt- t0) where Xt and X0 are cell density in cells/mL at specified time and initiation. Biomass, calculated as area under growth curve using cell densities. No-observed-effect-concentrations (NOEC) were determined using Williams’ Test (if data met tests for homogeneity and normality) or using Kruskal-Wallis Test. EC50 values were empirically estimated for these data because no results showed significant effects.

Biological Observations: Mean cell density (standard deviation) × 104 cells/mL

 

Control

Dose 1

Dose 2

Dose 3

Dose 4

Dose 5

24 hours

3.5 (1.4)

4.3 (1.1)

3.8 (0.3)

3.7 (1.4)

3.7 (0.3)

2.8 (0.7)

48 hours

23 (4.1)

23 (2.6)

23 (3.6)

24 (4.4)

19 (2.9)

23 (4.5)

72 hours

96 (16)

108(5.3)

96 (9.5)

100(5.9)

98 (24)

94 (11)

Validity criteria fulfilled:
not specified
Conclusions:
No phytotoxic effects were observed for maleic anhydride in P. subcapitata up to the highest test concentration of 150 mg maleic anhydride/L (nominal concentration) after static exposure for 72 h.
Executive summary:

In a 72-h acute toxicity study, the freshwater alga Pseudokirchneriella subcapitata was exposed to maleic anhydride at nominal concentrations of 0, 9.4, 19, 38, 75, 150 under static conditions in accordance with OECD guideline 201. Microscopic observations showed no abnormal cells in any treatment. There were no compound related phytotoxic effects.

The 72-h NOEC and EC50 values based on growth were 150 mg/l and > 150 mg/L (nominal concentration), respectively. Analytical measurements at 72 hours showed non-detectable concentrations in the 0 to 38 mg/L treatments. Concentration in the highest treatment was 110 mg/L at 72 hours, so the arithmetic average concentration was 130 mg/L [150 mg/L and 100 mg/L at 0 and 72 hours, respectively].

This toxicity study is classified as acceptable and satisfies the guideline requirement for OECD 201 toxicity study.

Results synopsis

Test organism: Pseudokirchneriella subcapitata

Test type: Static

72 h EC50 (growth): > 150 mg/L (nominal concentration)

72 h NOEC (growth): 150 mg/L (nominal concentration)

72 h NOEC (grwoth): 130 mg/L (arithm. mean measured concentration)

72 h EC50 (biomass):  > 150 mg/L (nominal concentration)

72 h NOEC (biomass): 150 mg/L (nominal concentration)

Description of key information

In a GLP guideline study according to OECD 201 (Bopanna, 2010), the toxicity of maleic acid to Pseudokirchneriella subcapitata was determined after static exposure for 72 h. The 72-h EC10 and EC50 values based on growth rate were 11.80 and 74.35 mg maleic acid/L, respectively. No abnormalities were noted. Upon molecular weight correction the effects values correspond to EC10 = 10.44 mg maleic anhydride/L and EC50 = 65.78 mg maleic anhydride/L.

In a second GLP guideline study according to OECD 201 (Springborn Smithers Laboratories, 2002) conducted with maleic anhydride, no toxic effects to Pseudokirchneriella subcapitata were noted at concentrations up to 130 mg/L (mean measured concentration) after static exposure for 72-h.

The more sensitive results of Bopanna (2010) are carried forward as key values in the chemical risk assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:
65.78 mg/L
EC10 or NOEC for freshwater algae:
10.4 mg/L

Additional information

The key study with Pseudokirchneriella subcapitata revealed an EC50 of 74.32 mg/l with non-neutralised test solution, based on algal growth-rate (Bopanna, 2010). The use of pH-adjusted test solution showed a decrease in effects. As a worst-case approach the EC50 estimated from non-neutralized samples was taken for further assessment.

Two additional studies are available (Hüls, 1988; Knie et al., 1983) which were, however, disregarded in the chemical safety assessment due to significant methodological deficiencies. Hüls (1988) observed high bacterial growth during test, which negatively impacts the overall test result, while Knie et al. (1983) used an unsuitable test system (determination of O2 production in the non-standard test species Haematococcus pluvialis after 4-h exposure).