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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:
08 January 1996 to 11 January 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984
Deviations:
yes
Remarks:
, see principles of method if other than guideline
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
1992
Deviations:
yes
Remarks:
, see principles of method if other than guideline
Principles of method if other than guideline:
According to OECD Guideline 201 when non-toxic growth inhibition takes places, e.g. due to light absorbing materials, then such physical types of effects should be separated from toxic effects by modifying the test conditions. A new method was developed by RCC Ltd for the quantification of algicidal effects due to reduced light intensities. In this case, two experiments took place. Experiment A occurred according to guideline, when Experiment B used the glass dishes above the cylinders containing the colored test media with the same test item concentrations as in part A however without algae. The algae grew in test water without test substance in Erlenmeyer flasks below and thus the inhibition occurred only due to light absorption.
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: BG 3247/TV 6
- Expiration date of the lot/batch: May 01, 1996

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, in the dark
- Stability under test conditions: in water > 3 days
- Solubility of the test substance in the solvent/dispersant/vehicle/test medium: approximately 100 g/l
Analytical monitoring:
yes
Details on sampling:
For the analytical measurements of the test substance concentrations duplicate samples were taken at the start of the test from the freshly prepared test media (without algae) of all test concentrations and from the control. For the determination of the stability of the test substance under the test conditions, sufficient volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test (but without algae) and were sampled in duplicate at the end of the test (after the 72 hours test period). The concentrations of the test substance FAT 40400/A were measured in the duplicate test media samples from all test concentrations and both sampling dates (0 and 72 hours). All samples are kept stored at -20 °C.
Vehicle:
no
Details on test solutions:
Dosage and concentrations:
The test medium of the highest test substance concentration was prepared by dissolving the test substance in test water (100 mg/L). Adequate volumes of the intensively mixed test medium were added to test water to prepare the test media of the following nominal concentrations: 1.0, 3.2, 10, 32, and 100 mg test substance/L. Additionally, a control was tested in parallel (test water without addition of the test substance). The test media were prepared just before the start of the test.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
The test organism used for the study was Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Sammlung von Algenkulturen, Pflanzenphysiologisches lnstitut der Universitilt Gottingen, D-37073 Gottingen, F.R.G. The algae were grown in the laboratories of RCC under standardized conditions according to the OECD Guideline No. 201.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg/L as CaCO3
Test temperature:
23.8-24.0°C
pH:
7.9-9.2
Nominal and measured concentrations:
- Nominal concentrations: 1.0, 3.2, 10, 32, and 100 mg/L.
- Measured concentrations: in the range from 81 % to 89 % of nominal.
- Mean measured concentrations (average over all measurements per test concentration): in the range of 82 % to 88 % of nominal.
Details on test conditions:
The test was started (0 hours) by inoculation of a biomass of 10.000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up about 72 hours prior to the test at the same conditions as in the test. The test was performed in Erlenmeyer flasks (50 mL), each with 50 mL algal suspension, continuously stirred by magnetic stirrers, 3 flasks per test concentration and 6 flasks in the control. Each Erlenmeyer flask was placed in a black cylinder, coated inside with aluminium foil. The cylinders were covered with glass dishes, the dishes were covered with watch glass dishes to prevent evaporation.

The test included two experimental parts:

- Experimental part A:
The algae grew in test media with dissolved test substance in the Erlenmeyer flasks ( 5 test concentrations and a control). All glass dishes above the
cylinders contained untreated test water. Thus, the inhibition of algal growth in this experimental part was caused due to a real toxic effect of the test substance and in addition to the reduced light intensities in the coloured test media in the Erlenmeyer flasks.
- Experimental part B
In this experimental part the glass dishes above the cylinders contained the coloured test media with the same five test concentrations as in part A however without algae (3 replicates per test concentration). In the Erlenmeyer flasks below, the algae grew in test water without test substance (as in the control), however under changed light conditions due to the filter effect of the coloured test media in the glass dishes. Thus, the growth
inhibition in part B was caused due to light absorption only. The depth of the test media in the glass dishes was 20 mm, i.e. half the depth of the test media in the Erlenmeyer flasks, because the algae in the stirred test media stay in the statistical mean in this mean depth.
All flasks were incubated in temperature controlled water baths and continuously illuminated at a mean light intensity of about 8100 Lux (range 7600-8460 Lux). The light intensity was measured just before the start of the test below the coating cylinders at nine places in the area, where the Erlenmeyer flasks were placed in the test. This illumination was achieved by fluorescent tubes (universal white L 25, 36 W) installed above the algal flasks.

Counting and examination of algal cells:
Small test media samples (0.1 - 1.0 mL) were taken out of all flasks after 24, 48 and 72 hours of exposure and were not replaced. The algae cell densities in the samples were determined by counting with an electronic particle counter (AL CELLCOUNTER, Model 871, AL-Systeme, D-76149 Karlsruhe), three measurements per sample. In addition, a sample was taken from the control and from the test concentration of nominal 100 mg/L in experimental part A with reduced algal growth after a test period of 72 hours. The shape of the treated algal cells was microscopically examined and compared with the cells in the control.
Reference substance (positive control):
no
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:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: no toxic effect seen upto 100 mg/l concentration
Details on results:
-E xperimental part A:
the test substance had a statistically significantly inhibitory effect on the growth of Scenedesmus subspicatus after the exposure period of 72 hours first at the concentration of 3.2 mg test substance/L (results of a Dunnett-test, one-sided, a= 0.05). 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 1.0 mg test substance/L, since at this test concentration the mean growth rates of the algae were statistically not significantly lower than in the control. The EC-values were calculated for the algal biomass band the grow1h rate~ after 72 hours test duration. According to the reconunendations of the Ad-hoc meeting of experts on algal growth inhibition tests for coloured substances (Vienna, 30-31 January 1996) the EC-values of the growth rates should be used for the classification of coloured substances.
-E xperimental part B:
the algal growth inhibition by the pure light effect (the reduced light intensities in the coloured test media) was quantified. In this experimental part nearly the same inhibition effect on the algal growth was observed compared to experimental part A. Thus, the algal growth inhibition was obviously caused only by the pure light effect. In experimental part B (light effect only), the mean algal growth rate was even slightly but statistically significant lower than in the control at the lowest test substance concentration of 1.0 mg test substance/L. The EC-values in experimental part B were in the same magnitude as the corresponding EC-values in experimental part A
Reported statistics and error estimates:
The EbC 50 and EµC 50 (the concentrations of the test substance corresponding to 50 % inhibition of algal biomass respectively growth rate compared to the control), and the corresponding EC 10 and their 95%-confidence limits were calculated for both experimental parts by Probit Analysis.
For the determination of the LOEC and NOEC, the calculated mean growth rates "µ" at the test concentrations were tested on significant differences to the control values by Dunnett-tests.

Comparison between the results in experimental parts A and B:

According to the recommendations of the Ad-hoc meeting of experts on algal growth inhibition tests for coloured substances (Vienna, 30-31 January 1996) 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 percentage inhibition of the growth rate µA minus the percentage inhibition of the growth rate µB after the 72 hours test period. At all test concentrations these differences were lower than 10%. As another measure of difference the quotient of the growth rates µA/µB was calculated for each test concentration. These quotients were also high (at least 0.9) at all test concentrations.

Differences in growth rates up to the magnitude of 10 % are accepted to be caused by pure chance in the used algal toxicity test. Thus, according to the reconunendations of the Adhoc meeting of experts on algal growth inhibition tests for coloured substances (Vienna, 30-

31 January 1996) the differences between inhibition in experimental part A and B should be lower than 10%, respectively the quotient µA/µB should be at least 0.9 or higher to accept that the inhibition curves of the growth rates µA and µB are essentially the same.

At all test concentrations of this test the differences of the growth rates µA and µB are lower than 10% and the quotients µA/ µB are at least 0.9.

General results

In the control the cell density has increased from nominal N = 1.0E+04 cells/mL at the start of the test (0 hours) to N= 71.7E+04 cells/mL (mean value) after 72 hours by a factor of approximately 72. Thus, the algal growth in the control was sufficiently high.

At the start of the test, the pH-values in test media were in the range of pH 7.9 - 8.0, at the end of the test pH-values were measured between pH 8.2 - 9.2. This increase of the pH-values was obviously caused by the CO2-consumption of algae during their rapid growth respectively their high densities (although the test flasks have been intensively stirred).

Validity criteria fulfilled:
yes
Conclusions:
A toxic effect of the test substance on the growth of Scenedesmus subspicatus can be excluded up to the highest test concentration of 100 mg test substance/l; so EC50 and NOEC of FAT 40400/A is > 100 and 100 mg/l respectively.
Executive summary:

The influence of the test substance on the growth of the green alga Scenedesmus subspicatus CHODAT was investigated in a 72-hour static test according to the OECD Guideline No. 201 and EU method C.3. However, the test method was modified to quantify the algicidal effect of the test substance, but also the growth inhibition effect due to reduced light intensities in the coloured test media. The test was performed in compliance with Good Laboratory Practice Regulations.

The nominal test concentrations were 1.0, 3.2, 10, 32 and 100 mg test substance/L and a control. All test media down to the lowest test concentration were slightly to strongly coloured by the test substance. The analytically determined test substance concentrations in the analysed test media varied in the range from 81 % to 89 % of the nominal values. The mean measured concentrations (calculated as the average over all measurements per test concentration) varied in the range of 82 % to 88 % of nominal. The test substance was sufficiently stable in the test media under the test conditions during the test period of 72 hours. Therefore, all biological results are related to the nominal concentrations of the test substance.

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

Thus, in conclusion, this modified algal test has clearly demonstrated that the observed growth inhibition effect of the test substance on Scenedesmus subspicatus was caused only due to an indirect effect, the light filter effect in the coloured test solutions.

Thus, a toxic effect of the test substance on the growth of Scenedesmus subspicatus can be excluded up to the highest test concentration of 100 mg test substance/l; so EC50 and NOEC of FAT 40400/A is > 100 and 100 mg/l respectively.

Description of key information

The toxicity of the test substance towards aquatic algae was investigated in a GLP-compliant test according to OECD guideline 201 and EU method C.3. In this study, freshwater alga (Scenedesmus subspicatus) were exposed for 72 hours to nominal concentrations of 0 (control), 1.0, 3.2, 10.0, 32.0, and 100 mg test substance/L under static conditions. In addition, as all test media were slightly to strongly coloured depending on the test substance concentration, alga were exposed to medium without test substance under varying reduced light conditions. This was done to differentiate between a direct toxic effect on algal growth, as induced by the test substance, and an indirect effect on growth caused by light absorption of the coloured test substance in solution. The effects on algal growth were similar in both experimental set-ups which demonstrates that the observed effect on algal growth was attributable only to light absorption caused by colouring of the test solutions. Based on these findings, the 72-h EbC50 and ErC50 values are determined at >100 mg/L. The NOEC is 100 mg/L for both endpoints.

Key value for chemical safety assessment

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

Additional information