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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
25 - 28 Oct 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Water solubility (under test conditions): Insoluble
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fraction (WAF): A nominal 1000 mg/L stock solution was prepared in culture medium by the WAF method. The stock solution was prepared by stirring the test material in dilution water for 20 hours, followed by settling for 4 hours. The aqueous layer was then removed for testing. The test solutions were prepared by adding the 1000 mg/L stock solution to culture medium water in appropriate quantities to obtain the desired concentrations for the test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, Windermere Laboratory
- Age of inoculum (at test initiation): Pre-culture growing in exponential phase
- Method of cultivation: Temperature: 23 ± 2 °C, Illumination: 6000 - 10000 lux continuous white light, shaking: 200 rpm, culture medium: according to OECD giudelines. Sterile nutrient stock solution were prepared and added to the dilution water to obtain the culture medium.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
test start: 22.5
test end: 21.5
pH:
test start: 7.4 - 7.6
test end: 7.2 - 7.3
Nominal and measured concentrations:
Nominal: 0, 100, 180, 320, 560, 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 250 mL conical flasks filled with 100 mL test medium
- Initial cells density: 10000 cells/mL
- Control end cells density: 153.89 x 10 exp 4 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: Deionised water sterilised by autoclaving at 120°C for 30 minutes
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH and temperature were measured on the pooled replicates for each test and control solution immediately prior to initiating the test and at the end of the full 72 hour test period.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes, to 8.0 ± 0.2
- Photoperiod: Continuous light
- Light intensity and quality: 6000 - 10000 lux white light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density was determined at 24, 48 and 72 hours on a small volume removed from each replicate flask. The cell counts were made using a haemocytometer and microscope.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1000 mg/L
- Results used to determine the conditions for the definitive study: EC50 as being approx. 1000 mg/L.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: WAF loading rate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: WAF loading rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: WAF loading rate
Details on results:
- Exponential growth in the control (for algal test): Yes
Reported statistics and error estimates:
The inhibition of growth was calculated using both the biomass integral (area under the growth curve) and the growth rate method.
The EL50 values were estimated where possible using a logarithm-linear or logarithm-probit plot of concentration and percent growth inhibition.

The test substance showed to be of low toxicity to Pseudokirchneriella subcapitata. The inhibiton for the WAF loading rate of 1000 mg/L after 72h by biomass integral was 18% and by growth rate 2%. Therefore, the 72 hour EL50 was estimated as > 1000 mg/L using the biomass integral and also by growth rate calculation. Because of the serial dilution from the highest concentration (1000 mg/L), only the 1000 mg/L test concentration was valid and could be used for evaluation of toxicity of the test substance. Without chemical analytics of the diluted concentrations, no information about the exact test concentration could be stated.

Table 1: Cell density measurements (Mean initial cell density: 1 x 10 exp 4 cells/ml)

Concentration [mg/L]

Replicate No.

Cell density measurements [cells/mL x 10 exp 4]

24 hours

48 hours

72 hours

0

1

2

3

4

5

6

3.00

2.67

2.67

2.33

1.67

2.00

37.00

30.33

27.33

41.33

28.00

33.67

149.33

157.33

150.67

148.67

168.00

149.33

Mean

2.39

32.94

153.89

100

1

2

3

2.00

3.33

3.67

42.67

47.00

38.33

166.67

165.33

140.00

Mean

3.00

42.67

157.33

180

1

2

3

2.67

3.33

2.67

34.33

30.67

28.67

164.00

182.67

158.67

Mean

2.89

31.22

168.45

320

1

2

3

1.33

2.67

1.67

37.33

30.00

25.33

168.00

172.33

164.33

Mean

1.89

30.89

168.22

560

1

2

3

2.67

3.00

1.67

30.67

24.67

20.00

129.33

144.00

168.67

Mean

2.45

25.11

147.33

1000

1

2

3

2.33

1.33

1.67

22.33

24.00

19.67

140.00

136.00

136.67

Mean

1.78

22.00

137.56

 

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
start date 26-02-2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (no test substance purity mentioned, no detailed description of exprimental conditions, positive control not reported, raw data lacking).
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 23 (Aquatic Toxicity Testing of Difficult Substances and Mixtures)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Water Accommodated Fraction (WAF) method: A aqueous solution was saturated with test substance and was stirred moderately for 68 hours by magnetic stirring with a 4 hours of settling period afterwards. The aqueous phase containing WAF was used to perform the test.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Source (laboratory, culture collection): Microbiotests Inc., Nazareth, Belgium
- The algae were prepared and conditioned by the supplier in alginate beads and were stored at 4 ± 3 °C until use
- Lot number: SC131107
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
none
Hardness:
no data
Test temperature:
23 ± 2 °C
pH:
Test begin: 8.1 ± 0.2
Test end: 8.9 in control, 9.4 in testing solution
Dissolved oxygen:
no data
Nominal and measured concentrations:
WAF containing 1.011 g/L
Details on test conditions:
TEST SYSTEM
- Initial cells density: 10000 cells/mL
- Control end cells density: 2794985 cells/mL

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD 201

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continued light
- Light intensity and quality: lateral lighting (white continued light, with spectra from 400 nm to 700 nm and intensity about 8000 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): growth inhibition after 24, 48 and 72 h
- Determination of cell concentrations: Measurement of the optical density at 670 nm, which was checked by counting with microscope.
Reference substance (positive control):
not specified
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: WAF loading rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopical examination of the algal cells at test ending confirmed the identity of the organism and revealed that the cells were normal in the test solution (i.e., no cell discoloration was noticed).
- The EL50 (72 h) was determined as > 1000 mg/L. The highest observed algae growth inhibition was 15.5%. Overall, the mean algae growth inhibition was 7.9%.
- The test item did not produce any particular effect during testing. No technical problem occurred during testing.
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
no data
Validity criteria fulfilled:
yes
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Feb - 02 Mar 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Water Accomodated Fraction (WAF): A concentration of 100 mg/L of the test substance was moderately stirred in algal medium for 24 h at room temperature. After this incubation time, the undissolved materials were removed by sterile filtration (0.45 µm) to obtain a sterile test substance solution for the algal growth test. The resulting water accommodated fraction was used in the test.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: 86.81 SAG
- Source: obtained from the collection of Algal Cultures, Institute of Freshwater Ecology, University of Göttingen, Göttingen, Germany
Age of inoculum (at test initiation): Exponentially growing liquid preculture were used to inoculate sterile test flasks.
- Method of cultivation: 250 mL flask containing 100 mL of sterile OECD medium inoculated with cell material from axenic slope culture, continuous illumination from warm white fluorescent tubes (8500 - 9000 lux), temperature: 22 ± 0.2 °C thermocontrolled room, periodically conducted reference test with potassium dichromate.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
not stated
Test temperature:
22 ± 0.2 °C
pH:
8.0 ± 0.2
Nominal and measured concentrations:
Nominal: 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Material: glass; Size: 250 mL; Fill volume: 100 mL
- Initial cells density: 10000 - 20000/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 5

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was determined in a control flask at the start and at the end of the test. The pH was determined in 1 control flask after sterilization before the test and in all test vessels at the end of the test. Observations of mis-shapen cells and cell-debris were made after 24, 48 and 72 h test duration.

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: warm white fluorescent tubes (8500-9000 lux)

EFFECT PARAMETERS MEASURED: Algal growth was recorded after 24, 48 and 72 h test duration.
- Determination of cell concentrations: using a spectrophotometer (680 nm wavelength). Aliquots of 5 mL were removed from each test substance served as controls. The starting cell density was determined using a counting chamber.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: EL50 (24-72h)
Key result
Duration:
72 h
Dose descriptor:
EL0
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: EL0 (24-72h)
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: EL50 (24-72h)
Key result
Duration:
72 h
Dose descriptor:
EL0
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: EL0 (24-72h)
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: no

At the WAF loading rate of 100 mg/L no inhibition with respect to algal growth rate and algal cell growth (area under growth curve) compared to the untreated controls were observed. The no-observed-effect concentration for growth rates (ErL0) and biomass production (EbL0) with respect to the loading rate was ≥ 100 mg/L. The EL50 -value was conducted as EL50 (24 -72 h). But the EL50 can also be stated as EL50 (72 h) > 100 mg/L for the algal growth rate and algal cell growth.

Table 1: Cell concentration (A680) in individual test cultures after 24, 48 and 72 h of exposure to the test material. ((b) = Mean absorbance (A680))

Nominal concentration [mg/L]

Code

Cell concentration (A680)

0 h (b)

24 h

48 h

72 h

Control

A

B

C

D

E

0.008

0.008

0.008

0.008

0.008

0.033

0.038

0.038

0.036

0.040

0.228

0.222

0.208

0.212

0.233

0.685

0.733

0.676

0.624

0.694

100

A

B

C

0.008

0.008

0.008

0.040

0.038

0.044

0.224

0.207

0.207

0.700

0.631

0.675

 

Description of key information

No relevant studies are available for decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane. Three algae toxicity studies are available for closely related structural analogues (CAS 11138-60-6 and CAS 91050-88-3).

Key value for chemical safety assessment

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

Additional information

Three algae toxicity studies are available for closely related structural analogues:

The first study with CAS 11138-60-6 conducted in accordance with OECD Guideline 201 determined a no-observed-effect concentration for growth rate (ErL0) of ≥ 100 mg/L and an ErL50 > 100 mg/L (WAF loading rate).

In the second study with CAS 91050-88-3 conducted in accordance with OECD Guideline 201, the 72h EL50 (WAF loading rate, based on growth rate) was determined to be > 1000 mg/L.

In the third study with CAS 11138-60-6 conducted in accordance with OECD Guideline 201, the 72h EL50 (WAF loading rate, based on growth rate) was determined to be > 1000 mg/L.

Therefore, by read-across, decanoic acid, mixed esters with heptanoic acid, octanoic acid and trimethylolpropane is not considered to be toxic to aquatic algae.

A report justifying the read-across approach is included in IUCLID Chapter 13.