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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 March 2006 to 26 June 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Deviations:
no
Principles of method if other than guideline:
However, the test method was modified following a method developed by RCC Ltd to quantify not only the algicidal effect of colored test substances, but also the growth inhibition effect caused by reduced light intensities in the colored test media.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identity: FAT 40824/A
Batch: Red ROE 805 BOP 04/05
Appearance: dark red powder
Purity: Organic part (Na-salt): approx. 82 %; Main component 1 : approx. 36.2 %; Main component 2: approx. 27.5 %; Oligomers: 10%
Expiration date: 01 October 2010
Stability in water: Max. 7 days at room temperature
Solubility in water: >100g/Lat20°C
Storage: At room temperature at about 20 °C, in a desiccator because test substance is hygroscopic, away from direct sunlight
Analytical monitoring:
yes
Details on sampling:
Before the start of the test and after 72 hours, duplicate samples from each test medium and the control (without algae).
All samples were deep-frozen (at about -20 °C) immediately after sampling.
Vehicle:
no
Details on test solutions:
The following concentrations were tested: nominal 4.6, 10, 22, 46 and 100 mg/L. Additionally, a control was tested in parallel.
The test medium of the highest test item concentration of nominal 100 mg/L was prepared by dissolving 82.4 mg of the test item completely in 800 mL of test water using stirring (15 minutes at room temperature). Adequate volumes of this intensively mixed test medium were diluted with test water to prepare the test media with the lower test item concentrations. The test media were prepared just before addition of algae.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
The algal cells were taken from an exponentially growing pre-culture which was set up three days prior to the test under the same conditions as in the test. The test organism used for the study was Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, D-37073 Göttingen, Germany). The algae had been grown in RCC's laboratories under standardized conditions according to the test guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg/L as CaCO3
Test temperature:
23 - 24 °C
pH:
Start: 7.9 - 8.1
End: part A: 8.2 - 9.0
part B: 8.6 - 9.4
control: 9.2
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Nominal: 4.6, 10, 22, 46 and 100 mg/L
Measured: between 89 and 95% of the nominal values
Details on test conditions:
Inoculation: 10,000 algal cells per mL of test medium.
Vessels: erlenmeyer flasks 50 mL
Volume: 15 mL algal suspension per flask
Parallel: 3 flasks per concentration, 6 flasks per control
Light condition: continuously illuminated about 8500 Lux (mean value), range:7040 to 9600 Lux
The flasks were continuously stirred by magnetic stirrers.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: the results of experimental part A, 95%-confidence limits was 37-59.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: the results of experimental part A
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: the results of experimental part A
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: the results of experimental part A
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
62 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: the results of experimental part B, 95%-confidence limits was 38-156
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: the results of experimental part B
Details on results:
In experimental part B, a very similar inhibitory effect on the algal growth was observed compared to experimental part A.
The EC values in experimental part B were in the same magnitude as the corresponding EC values in experimental part A.
Results with reference substance (positive control):
No data
Reported statistics and error estimates:
Inhibition of algal growth was determined from the area under the growth curves AUC and the specific growth rates (r) for exponentially growing cultures.

The microscopic examination of the algal cells after 72 hours exposure showed there were no obvious effects on the shape and size of the algal cells growing in test media containing the test item at up to and including 100 mg/L concentration.

The analytically determined concentrations of the test item in the test media were between 89 and 95% of the nominal values at the start and the end of the test. The test item was stable during the test period of 72 hours under the test conditions. The biological results were based on the nominal concentrations of the test item.

Experimental part A

Experimental part A corresponds to the usual algal toxicity test. This means that the algal growth inhibition in this experimental part was caused by a possible toxic effect of the test item and/or by the reduced light intensities due to the light absorption in the coloured test media. In experimental part A, the test item had a statistically significant inhibitory effect on the growth rate r of Scenedesmus subspicatus after the exposure period of 72 hours first at the concentration of 10 mg/L (results of a Dunnett-test, one-sided, a = 0.05, Table 5). Thus, this test concentration was determined as the 72-hour LOEC (lowest concentration tested with toxic effects). The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours) was determined at the concentration of 4.6 mg/L, since up to and including this test concentration the mean growth rate r of the algae was statistically not significantly lower than in the control. For the biomass b, the same LOEC and NOEC values were determined.

The microscopic examination of the algal cells after 72 hours exposure showed no difference between the algae growing in test medium with reduced algal growth (nominal 100 mg/L) in experimental part A and the algal cells in the control. There were no obvious effects on the shape and size of the algal cells growing in test media containing the test item at up to and including this nominal concentration.

The EC values as well as the NOEC and LOEC were calculated for the algal biomass b and the growth rate r after 72 hours test duration:

Experimental part A

Parameter (0-72 h)

Biomass b (mg/L)

Growth rate r (mg/L)

EC50

95%-confidence limits

46

37-59

> 100

n.d.

EC10

95%-confidence limits

5.4

3.5-7.5

19

16- 22

EC90

95%-confidence limits

>100

n.d.

>100

n.d.

NOEC

4.6

4.6

LOEC

10

10

 

Experimental part B

In experimental part B, the algal growth inhibition caused by the pure light effect (the reduced light intensities in the coloured test media) was quantified. In this experimental part, a very similar inhibitory effect on the algal growth was observed compared to experimental part A.

The EC values in experimental part B were in the same magnitude as the corresponding EC values in experimental part A:

 

Experimental part B

Parameter (0-72 h)

Biomass b (mg/L)

Growth rate r (mg/L)

EC5O

95%-confidence limits

62

38-156

> 100

n.d.

EC10

95%-confidence limits

15

3.7-25

34

16-55

 

Comparison between the results in experimental parts A and B

According to the recommendations of the Ad-hoc working group of experts on algal growth inhibition for the interpretation of test results of coloured substances, the comparison between the results in experimental parts A and B was based on the growth rates.

The differences between the results of experimental parts A and B were described for each test concentration as the percentage inhibition of the growth rate rA (lrA) minus the percentage inhibition of the growth rate rB (lrß) after the 72 hours test period.

As another measure of difference, the quotient of the growth rates rA/rB was calculated for each test concentration.

Differences in growth rates up to the magnitude of 10% are accepted to be caused by pure chance in the algal toxicity test. Thus, according to the above mentioned recommendations of the Ad-hoc working group of experts, the difference between inhibition in experimental part A and B should be not higher than 10%, and the quotient rA/rB should be at least 0.9 or higher in order to assume that the inhibition curves of the growth rates rA and rB are essentially the same.

At all test concentrations in this test, the difference between the inhibition of growth rates rA and rB was lower than 10% (Table 4 and Figure 1), and the quotient rA/rB was at least 0.9.

Validity criteria fulfilled:
yes
Conclusions:
This modified algal test has clearly demonstrated that the observed growth inhibition effect of the test item FAT 40824/A on Scenedesmus subspicatus was caused only by an indirect effect, the light filter effect in the colored test solutions. Thus, a toxic effect of the test item on the algal cells can be excluded up to the highest test concentration of 100 mg/L.
Executive summary:

In a GLP compliant study, the influence of the test item FAT 40824/A on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, Annex Part C.3, 1992, and the OECD Guideline No. 201, 1984. However, the test method was modified to quantify not only the algicidal effect of the test item, but also the growth inhibition effect caused by reduced light intensities in the colored test solutions. The nominal test concentrations were 4.6, 10, 22, 46 and 100 mg/L in parallel with a control. All test media down to the lowest test concentration were slightly to strongly colored by the test item.

The analytically determined concentrations of the test item in the test media were between 89 and 95% of the nominal values at the start and the end of the test. The test item was stable during the test period of 72 hours under the test conditions. The biological results were based on the nominal concentrations of the test item.

The test design consisted of two experimental parts. In experimental part A, the algae were grown in the presence of different test item concentrations as in the usual algal toxicity test. In experimental part B, the algal growth inhibition caused by the pure light effect (the reduced light intensities in the colored test media) was quantified: the algae grew in test water without test item (as in the control), however under changed light conditions due to the filter effect of the colored test media in glass dishes above the algal suspensions. These dishes contained test medium with same test item concentrations as in part A, however without algae. The results in experimental parts A and B based on growth rates were compared.

The same growth inhibition of Scenedesmus subspicatus was observed when the algae grew in test water without test item, but under reduced light intensities by the filter effect of the colored test media as in the second parallel experimental part, where the algae grew in the test media with dissolved test item.

 

In conclusion, this modified algal test has clearly demonstrated that the observed growth inhibition effect of the test item FAT 40824/A on Scenedesmus subspicatus was caused only by an indirect effect, the light filter effect in the colored test solutions. Thus, a toxic effect of the test item on the algal cells can be excluded up to the highest test concentration of 100 mg/L.

Description of key information

Results for acute toxicity test to green algae (72 hours exposure): EC10 and EC50: ≥100 mg/l

Key value for chemical safety assessment

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

Additional information

In a GLP compliant study, the influence of the test item FAT 40824/A on the growth of the green algal species Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, Annex Part C.3, 1992, and the OECD Guideline No. 201, 1984. However, the test method was modified to quantify not only the algicidal effect of the test item, but also the growth inhibition effect caused by reduced light intensities in the colored test solutions. The nominal test concentrations were 4.6, 10, 22, 46 and 100 mg/L in parallel with a control. All test media down to the lowest test concentration were slightly to strongly colored by the test item.

The same growth inhibition of Scenedesmus subspicatus was observed when the algae grew in test water without test item, but under reduced light intensities by the filter effect of the colored test media as in the second parallel experimental part, where the algae grew in the test media with dissolved test item.

 

In conclusion, this modified algal test has clearly demonstrated that the observed growth inhibition effect of the test item FAT 40824/A on Scenedesmus subspicatus was caused only by an indirect effect, the light filter effect in the colored test solutions. Thus, a toxic effect of the test item on the algal cells can be excluded up to the highest test concentration of 100 mg/L.