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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:
May 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
Principles of method if other than guideline:
The study also followed the Guidance document on aquatic toxicity testing of difficult test substances and mixtures. OECD series on testing and assessment number 23, December 14, 2000.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Final test: A water soluble fraction (WSF) prepared as a supernatant of an ultra-centrifuged dispersion with a nominal loading rate of 100 mg/l (100%) and a range of subsequent dilutions containing 10, 18, 32 and 56%.

- Sampling method:
Range-finding test: Samples for possible analysis were taken from all concentrations.
Limit test: Samples for chemical analysis were taken at the start and the end of the test.
Final test: During the final test no samples for analysis were taken as in either of the previous tests chemical analysis showed no detectable levels of the test substance in any of the samples taken from the water soluble fraction tested.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- The batch of EA-3098 tested was a white powder containing a mixture of components. The water solubility of these components was at the highest less than 1.0 x 10-4 g/l. Preparation of test solutions started with weighed amounts of test substance that were quantitatively added to volumetric flasks. In the range-finding test the solutions were treated with ultra-sonic waves for 10 minutes and then stirred for 30 minutes prior to filtration (0.45 µm, Schleicher and Schuell). The final test solutions were all clear and colourless.

in the limit and the final test, preparation started with quantitatively adding weighed amounts of test substance to Milli-Q water. These solutions were then stirred for 72 hours. subsequently they were ultra-centrifuged at relative centrifugal fields of up to 108,800 g and the salts for the ISO-medium (M2) were added to the supernatents to obtain the final test solutions. The final test solutions were all clear and colourless.

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 10E4 cells/ml.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: Range-finding test: CCAP 278/4. Final test: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation:
Stock culture: algal stock cultures were started by inoculating growth medium with algal cells from a pure culture in agar. the suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 +/- 2°C.
Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2.1E+04 cells/ml. The pre-culture was maintained under the same conditins as used in the test. The cell density was measured immediately before use.

ACCLIMATION
- Acclimation period: not stated in report
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: not applicable
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable.

(All test and control cultures were inspected microscopically at 72 hours).
Hardness:
24 mg CaCO3/l
Test temperature:
Temperature was monitored continuously in a temperature control vessel.

23 +/1 °C
pH:
pH was determined at the beginning and end of the test.

start: 7.9 - 8.0
end: 8.0 - 9.6
Dissolved oxygen:
Not stated in report
Salinity:
Not applicable as freshwater study
Nominal and measured concentrations:
Nominal: 0, 10, 18, 32, 56 and 100 mg/l water soluble fraction (WSF)
Measured: Measured concentrations of all three peaks were below their limits of detection.
Details on test conditions:
STOCK CULTURE MEDIUM:
M1; formulated using Milli-Ro water (tap-water purified by reverse osmosis).

PRE-CULTURE MEDIUM:
M2, according to ISO-Standard 8692, formulated using Milli-Q water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges, preventing precipitation.

RANGE-FINDING TEST:
A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
- The solutions were treated with ultra-sonic waves for 10 minutes and then stirred for 30 minutes before filtering through a 0.45 µm filter.
- Exponentially growing algal cultures were exposed to solutions containing 0, 0.1, 1, 10 and 100% of the filtrate prepared at 100 mg/l.
- Three replicates were tested per concentration and three replicates in the control group. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.
- pH was only measured in the control and the highest test concentration. Samples for possible analysis were taken from all concentrations.

LIMIT TEST:
The project continued with a limit test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
Six replicates of exponentially growing algae were exposed to a water soluble fraction prepared as a supernatant of an ultra-centrifuged dispersion (48,000 x g) with a nominal loading rate of 100 mg/l or an untreated control. Samples for chemical analysis were taken at the start and the end of the test.

FINAL TEST:
Test concentrations:
EA-3098: A water-soluble fraction (WSF) prepared as a supernatant of an ultra-centrifuged dispersion (108,800 x g) with a nominal loading rate of 100 mg/l (100%) and a range of subsequent dilutions containing 10, 18, 32 and 56 %.

Controls: Test medium without test substance or other additives (blank-control).

Replicates:
3 replicates of each test concentration
6 replicates of the blank control
1 replicate of the highest concentration without algae.

Test procedures and conditions:
Test duration: 72 h
Test type: Static
Test vessels: 100 ml, all-glass, containing 50 ml of test solution.
Medium: M2
Cell density: An initial cell density of 1 x 10E4 cells/ml.
Illumination: Continously using TLD-lamps of the type 'cool-white' of 30 Watt, with a light intensity within the range og 78 to 92 µE.m-2.s-1
Incubation: Vessels were distributed at random in the incubator. During incubation the algal cells were kept in suspension by continous shaking.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

pH: At the beginning and at the end of the test. The pH of the solutions should preferably not deviate by more than 1.5 units during the test.
Temperature of medium: Continously in a temperature control vessel.

RECORDING OF CELL DENSITIES:
At the beginning of the test. cells were counted using a microscope and a counting chamber.
Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank.





















Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 18 - <= 32 other: % of water soluble fraction
Nominal / measured:
nominal
Conc. based on:
other: water soluble fraction (WSF)
Basis for effect:
growth rate
Remarks on result:
other: EC50 between 18 and 32% of water soluble fraction (WSF)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 other: % of water soluble fraction
Nominal / measured:
nominal
Conc. based on:
other: water soluble fraction (WSF)
Basis for effect:
growth rate
Remarks on result:
other: The NOEC of EA-3098 for growth rate reduction corresponded with 10% of its water soluble fraction (WSF)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 18 - <= 32 other: % water soluble fracttion
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
water soluble fraction (WSF)
Basis for effect:
cell number
Remarks on result:
other: EC50 between 18 and 32% of water soluble fraction (WSF)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
18 other: % water soluble fraction
Nominal / measured:
nominal
Conc. based on:
other: Water soluble fraction (WSF)
Basis for effect:
cell number
Remarks on result:
other: The NOEC of EA-3098 for cell growth inhibition corresponded with 18 % of its water-soluble fraction.
Details on results:
RANGE-FINDING TEST:
Mean cell densities, inhibition of cell growth and reduction of growth rate:
Chemical analysis revealed that concentrations of all three major compounds in the WSF prepared at 100 mg/l were below their limits of detection.9 (Tables 4- 6 of the Analytical report - see attached background material).

All test conditions were maintained within the limits prescribed by the protocol except for pH which exceeded the upper limit of 9.0. Since the effects on total cell growth and growth rate were below 50%, the expected EC50-values were above the maximum solubility level of EA3098 in water. However, the test solutions were only stirred for 30 minutes. Since no detectable concentrations were found it was decided to stir for 72 h instead. This period was considered to be sufficient to reach a saturated solution. Further it was decided to use ultra-centrifugation instead of filtration through a 0.45 µm filter to exclude possible adsorption of the dissolved components of EA-3098 to the filter.

LIMIT TEST:
Mean cell densities, inhibition of cell growth and reduction of growth rate:
Again chemical analysis revealed that concentrations of all three major compounds WSF prepared at 100 mg/I were below their limits of detection (Tables 7 - 9 of the Analytical Report). Hence, no detectable concentrations were present in spite of prolonging the stirring period to 72 h and using centrifugation Instead of filtration.

All test conditions were maintained within the limits prescribed by the protocol. The effect of exposure to the WSF of EA-3098 was slightly higher compared to the range-finding test. In order to be able to calculate ECso-values it was decided to perform an additional test with a range of dilutions of the WSF of EA-3098.

FINAL TEST:
Mean cell densities:
Respective growth curves are shown in Figure 1 (see attached background material). Also see Appendix 1 (in attached background material) for individual cell densities and growth rate.
Contrary to the previous tests, no signficant growth of algal cells was recorded in the WSF prepared at 100 mg/l or in the dilutions containing 32 and 56%.
Inhibition of cell growth and reduction of growth rate:
Complete inhibition of cell growth and growth rate were recorded in 32 to 100% of the WSF. No significant differences were recorded between the values for cell growth inhibition in the blank control and the two lowest dilutions containing 10 and 18% of the WSF. Growth rate was significantly affected at 18% of the WSF for the 0-24 h and 0 -48 h intervals, but not for the whole test period.

DETERMINATION OF EFFECT CONCENTRATIONS:
No relation could be made with chemically identified components as none of the three major components were detected.

EXPERIMENTAL CONDITIONS:
THe pH did not remain within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit). The pH exceeded the upper limit at the end of the test in the blank-control and the solutions containing 10 or 18% of the WSF. THe algal growth in these vessels increased by a factor of >100, which caused this increase in pH due to CO2 consumption. However, at the time this would affect further algal growth the test was already ended. The temperature of the test medium was 22.2°C at the start of the test. During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the protocol (21-25°C, constant within 2°C).

ACCEPTABILITY OF THE TEST:
1) In the controls, cell density increased by an average factor of > 16 within 3 days.
2) Further, test conditions (temperature and pH) remained within the ranges prescribed by the guideline, except for the pH in the control and the two lowest treatment levels. The pH increased above 9.0 and by more than 1.5 unit, but this was related to a relative high rate of algal growth. Hence, this deviation did not affect the validity criterion for algal growth.












Results with reference substance (positive control):
- Results with reference substance valid? Yes

- EC50: For cell growth inhibition = 0.63 mg/l. For growth rate reduction = 1.3 mg/l

Final Test:

Pecentage inhibition of cell growth during the final test

Concentration EA-3098: % of a WSF prepared at 100 mg/l

Cell growth (0-72 h)

Mean Area (A)

Inhibition (%)

0

2811

 

10

2448

12.9

18

2346

16.5

32

0

100

56

0

100

100

0

100

Determination of effect concentrations:

The table below shows the effect parameters of EA-3098 expressed as % of a WSF prepared at 100 mg/l.

Parameter

Concentration EA-3098: % of a WSF prepared at 100 mg/l (= solubility level: <0.1 mg/l)

NOEBC

18

72 h EBC50

Between 18 and 32

NOERC

10

72 h ERC50

Between 18 and 32

Under the conditions of the tests conducted with Selenastrum capricornutum, EA-3098 reduced growth rate of this fresh water algae species significantly at its water-soluble fraction. The water solubility of the major components was all below their detection levels. Hence, the complete inhibition found in the final test indicated the presence of a possible algaecidal component or degradation product that could not be identified with the analytical method available.

The difference in response of algae tested in the range-finding and limit compared to those tested in the final test was related to the difference in algal string used. The NIVA CHL 1 string has proven to be more sensitive than the CCAP 278/4 string

The EC50 of EA-3098 for both cell growth inhibition and growth rate reduction were between 18 and 32 % of its water-soluble fraction.

The NOEC of EA-3098 for cell growth inhibition corresponded with 18 % of its water-soluble fraction. The NOEC of EA-3098 for growth rate reduction corresponded with 10 % of its water soluble fraction.

Validity criteria fulfilled:
yes
Conclusions:
The EC50 of the test material for both cell growth inhibition and growth rate reduction were between 18 and 32% of its water-soluble fraction.

The NOEC of the test material for cell growth inhibition corresponded with 18% of its water-soluble fraction. The NOEC of EA-3098 for growth rate reduction corresponded with 10% if its water soluble fraction.
Executive summary:

The study procedures used were based on OECD guideline No. 201. In addition, the procedures were designed to meet the test methods of EEC directive 62/69 part C.3 and ISO International Standard 8692.

The batch of test material tested was a white powder containing a mixture of components. The water solubility of the major components was at the highest <1.0 x 10-4 g/l. Preparation of test solutions was aimed at a water soluble fraction (WSF), which included stirring for 30 minutes followed by ultra-filtration (range-finding test) or 72 hours followed by ultra-centrifugation (limit and final test). The final test solutions were all clear and colourless. After preparation, volumes of 50 ml and 1 ml of algal suspension were added to each replicate of the respective test concentration (initial cell density of 104 cells/ml). The total exposure period in each test was 72 hours.

In a range-finding test chemical analysis revealed that concentrations of all major compounds in the WSF prepared at 100 mg/l were below the limits of detection. Based on the results of the range-finding test the expected EC50 values were above the maximum solubility level of the test material in water. a limit test was performed stirring a 100 mg/l dispersion fo 72 hours, followed by ultra-centrifugation instead of filtration to exclude possible adsorption of the dissoled components of the test material to the filter. The effect of exposure to the WSF of the test material was slightly higher compared to the range-finding test.

The project continued with a full test including a range of dilutions of the WSF prepared at 100 mg/l, but without analytical support as no detectable levels of any of the components of the test material were expected. The test included 3 replicates of each dilution and 6 replicates of the blank-control. Contrary to the earlier tests, no significant growth of algal cells were recorded in the WSF prepared at 100 mg/l or in the dilutions containing 32 and 56%. As a result, complete inhibition of cell growth and growth rate were recorded in 32 to 100% of the WSF. No significant cell growth inhibition was recorded in the two lowest dilutions containing 10 and 18% of the WSF, whereas growth rate was significantly affected at 18% for the 0 -24 and 0 -48 hour intervals, but not for the whole test period.

In conclusion, the test material reduced the growth rate of Selenastrum capricornutum significantly at its WSF. The water solubility of the major components were all below their detection levels. The analytical support did not detect concentrations of the major polymeric compounds related to the test material, while the original monomers were not measured by the analytical method used. Hence, the complete inhibition found in the final test indicated the presence of a possible algaecidal component or degradation product that could not be identified with the analytical method available.

The EC50 of the test material for both cell growth inhibition and growth rate reduction were between 18 and 32% of its water soluble fraction (WSF)

The NOEC of the test material for cell growth inhibition corresponded with 18% of its WSF. The NOEC of the test material for growth rate reduction corresponded with 10% of its WSF.

Description of key information

The test material reduced the growth rate of Selenastrum capricornutum significantly at its water soluble fraction. The water solubility of the major components were all below their detection levels.

The EC50 of the test material for both cell growth inhibition and growth rate reduction were between 18 and 32% of its water soluble fraction (WSF)

The NOEC of the test material for cell growth inhibition corresponded with 18% of its WSF. The NOEC of the test material for growth rate reduction corresponded with 10% of its WSF.

Key value for chemical safety assessment

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

Additional information

The study procedures used were based on OECD guideline No. 201. In addition, the procedures were designed to meet the test methods of EEC directive 62/69 part C.3 and ISO International Standard 8692.

The batch of test material tested was a white powder containing a mixture of components. The water solubility of the major components was at the highest <1.0 x 10-4g/l. Preparation of test solutions was aimed at a water soluble fraction (WSF), which included stirring for 30 minutes followed by ultra-filtration (range-finding test) or 72 hours followed by ultra-centrifugation (limit and final test). The final test solutions were all clear and colourless. After preparation, volumes of 50 ml and 1 ml of algal suspension were added to each replicate of the respective test concentration (initial cell density of 104cells/ml). The total exposure period in each test was 72 hours.

In a range-finding test chemical analysis revealed that concentrations of all major compounds in the WSF prepared at 100 mg/l were below the limits of detection. Based on the results of the range-finding test the expected EC50 values were above the maximum solubility level of the test material in water. A limit test was performed stirring a 100 mg/l dispersion for 72 hours, followed by ultra-centrifugation instead of filtration to exclude possible adsorption of the dissolved components of the test material to the filter. The effect of exposure to the WSF of the test material was slightly higher compared to the range-finding test.

The project continued with a full test including a range of dilutions of the WSF prepared at 100 mg/l, but without analytical support as no detectable levels of any of the components of the test material were expected. The test included 3 replicates of each dilution and 6 replicates of the blank-control. Contrary to the earlier tests, no significant growth of algal cells were recorded in the WSF prepared at 100 mg/l or in the dilutions containing 32 and 56%. As a result, complete inhibition of cell growth and growth rate were recorded in 32 to 100% of the WSF. No significant cell growth inhibition was recorded in the two lowest dilutions containing 10 and 18% of the WSF, whereas growth rate was significantly affected at 18% for the 0 -24 and 0 -48 hour intervals, but not for the whole test period.

In conclusion, the test material reduced the growth rate of Selenastrum capricornutum significantly at its WSF. The water solubility of the major components were all below their detection levels. The analytical support did not detect concentrations of the major polymeric compounds related to the test material, while the original monomers were not measured by the analytical method used. Hence, the complete inhibition found in the final test indicated the presence of a possible algaecidal component or degradation product that could not be identified with the analytical method available.

The EC50 of the test material for both cell growth inhibition and growth rate reduction were between 18 and 32% of its water soluble fraction (WSF)

The NOEC of the test material for cell growth inhibition corresponded with 18% of its WSF. The NOEC of the test material for growth rate reduction corresponded with 10% of its WSF.