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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Validity criteria from the OECD 201 versions 1984 and 2006 were not met since the cell concentration in the controls hat increased only by a factor of 15.38 instead of the required factor of 16.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
light intensity too low (4400 Lux) instead of 6800 to 9600 Lux
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 8.1, 27, 90, 300, 1000 mg MRD-92-428 (CT-519-92S)/L
- Sampling method: Samples were removed from each treatment on days 0 and 3, filtered through a 0.45 µm Teflon filter and analysed for dissolved organic carbon content.
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Selenastrum capricornutum, new name Pseudiokirchneriella subcapitata
- Strain: # 1648
- Source (laboratory, culture collection): provided by the Department of Botany, University of Texas
- Age of inoculum (at test initiation): The algae were taken from the log phase of the culture
- Method of cultivation: similar to test conditions
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
23,2 +/- 0.2 °C
Details on test conditions:
TEST SYSTEM
- Test vessel: 125 mL Erlenmyer flasks, filled with 50 mL medium
- Type (delete if not applicable): test flasks were closed with cotton-gauze stoppers
- Initial cells density: 7.200 to 9.600 cells /mL
- Control end cells density: after 72 hours: 140.000 cells /mL; after 96 hours: 190.000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water
- Metals and pesticides were below level of concern
- Culture medium different from test medium: No
- Intervals of water quality measurement: initial measurement and at test termination

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: continuous
- Light intensity and quality: 4400 Lux
- Salinity (for marine algae):

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter (Turner filter fluorimeter)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: about 3.2
- Range finding study
- Test concentrations: control, 5, 10, 50, 100 and 1000 mg test item/L, in triplicate, 72 hours
- Results used to determine the conditions for the definitive study: A noticeable reduction in growth occurred at 100 and 1000 mg test item/L, slight inhibition was exhibited at 10 and 5 mg test item/L.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 960 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 960 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): no
- Any stimulation of growth found in any treatment: All treated test groups exhibited approximately equal or higher growth grates than the controls. After 72 hours these were statistically significantly higher at 7.78, and 86.4 mg a.i./L and after 96 hours at 7.78, 86.4 and 288 mg a.i./L. A slight inhibitory effect of 4.1 % of the growth rate was observed at 72 hours in the 25,92 mg a.i./L treatment level.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: none
Reported statistics and error estimates:
The data were evaluated using the ANOVA procedure of SAS for NOEC determination. For the EC50 determination, based on specific growth rate an inverse interpolation method of Snedecor and Cochran was used.

Table 1: Evaluation if the validity criteria of the updated OECD 201 (version 2006) were fulfilled.

NOTE: when the test was performed, the OECD 201 Testguideline from 7 June 1984 was in place.

Cell numbers in cells/mL
  Days   0-1d 1-2d 2-3d 0-3d Average SD CV
0 1 2 3  
Repl 1 9300 22000 79000 110000 µ= 0,86 1,28 0,33 0,82 0,82 0,47 57,66
Repl 2 8400 11000 60000 96000 µ= 0,27 1,70 0,47 0,81 0,81 0,77 95,12
Repl 3 8700 26000 120000 200000 µ= 1,09 1,53 0,51 1,04 1,04 0,51 48,91
Mean   19666,67 86333,33 135333,33 Mean 0,74 1,50 0,44 0,89
SD   11700,72 49901,54 81866,54 SD 0,43 0,21 0,09 0,13
1. Validity criterion: coefficient of variation of average specific growth rate during the whole test period in replicates control cultures:
Accepted 7 % by using Pseudokirchneriella(ex-Selenastrum) andDesmodesmus(ex-Scenedesmus), for less frequent tested species: 10 %
CV 0-1d 1-2d 2-3d 0-3d
59,50 57,80 60,49 14,70
Alternative calculation of the CV (OECD 201 does not mention the way how to calculate the CV):
                 
      0-1d 1-2d 2-3d      
    CV 57,33 14,01 21,55 30,97    
                 
Scientifically this alternative calculation is more reasonable. But as just a few studies will fullfil this criterion, this calculation should not be used for pracmatic reasons. 
2. Validity criterion: Coefficient of variation for section-by section growth rates (days 0-1; 1-2 and 2-3):
Replicates
according to OECD 201 < 35 %!  
  CV 67,23  
3. Validity criterion:Exponential Increase:
        According to OECD 201: Increase by the factor of at least 16 in 72 h          
after 72 h: 15,38 times increase after 48 h: 9,81 times increase
ln (Cell numbers):
0 1 2 3
Repl 1 9,13777 9,99879773 11,2772 11,608236
Repl 2 9,035987 9,30565055 11,0021 11,472103
Repl 3 9,071078 10,1658518 11,69525 12,206073
CV Average specific growth rate 14,70 % not fullfilled
CV- section-by section growth rate 67,23 % not fullfilled
Exponential Increase >16 times 15,38 times not fullfilled
Validity of the test according to OECD 201 (2006): not valid

Table 2 Mean cell concentration in the test groups

Concentration

Mean cell concentration [cells/mL]

nominal mg test item/L

nominal a.i./L

Day 0

Day 1

Day 2

Day 3

Day 4

Control

0

8800

20000

86000

140000

190000

8,10

7,78

9100

26000

68000

310000

890000

27

25,92

9000

20000

69000

180000

340000

90

86,4

7500

24000

120000

440000

970000

300

288

8500

16000

85000

230000

540000

1000

960

8700

16000

75000

120000

220000

Table 3 pH values in the test solution at test initiation and test termination (96 hours)

Concentration

pH

nominal mg test item/L

nominal a.i./L

Day 0

Day 4

Control

0

7.5

7.3

8.10

7.78

7.4

7.5

27

25.92

7.4

7.5

90

86.4

7.4

7.5

300

288

7.5

7.5

1000

960

7.6

7.4

Validity criteria fulfilled:
no
Remarks:
Within 72 hours, the cell concentration increased by a factor of 15.38 instead of 16 as described by the guideline (version 7 June 19984 and 23 March 2006).
Conclusions:
Even if the test does not fulfil the guideline validity criteria there it is good evidence provided that the ErC50 is > 960 mg a.i./L.
Executive summary:

In order to determine the growth of the cultures, the cell density was determined by fluorescence measurement. The cell density was determined at 0, 24, 48, 72 and 96 hours. The pH as measured at hour 0 and hour 96 ranged from 7.3 to 7.6. The growth of the control cultures does not fulfill the validity criteria from OECD 201 (1984 and 2006) since the cell concentration in the controls hat increased only by a factor of 15.38 instead of the required factor of 16. In addition the further control growth rate validity criteria outlined in the revised OECD 201 (version 23 March 2006) guideline were not met. Only in the 25.92 mg a.i./L test group a slight inhibition was observed after 72 hours. All other test groups showed similar or higher growth rates when compared to the control. There was no dose response relationship observed. Due to the lack of inhibitory effects, no NOEC was defined. Hence, based on the obtained data, the 72 -hour and 96-hour ErC50 is > 960 mg a.i./L.

This study is considered to be reliable with restrictions. The cell concentration increase was slightly below the validity criterium. However, the results were close to the trigger and hence the results may be regarded together with further information as relevant for the risk assessment.

Description of key information

ErC50 = > 960 mg a.i./L

Key value for chemical safety assessment

EC50 for freshwater algae:
960 mg/L

Additional information

For this endpoint there is one study from Cytec (Targia, 1993) available. beside some shortcomings in the performance of the control test group this GLP study is acceptable for the risk assessment (Klimisch score 2) and hence considered to be the key study.

In this study the cell density was determined at 0, 24, 48, 72 and 96 hours. The pH as measured at hour 0 and hour 96 ranged from 7.3 to 7.6. The growth of the control cultures does not fulfil the validity criteria from OECD 201 (1984 and 2006) since the cell concentration in the controls hat increased only by a factor of 15.38 instead of the required factor of 16. In addition the further control growth rate validity criteria outlined in the revised OECD 201 (version 23 March 2006) guideline were not met. Only in the 25.92 mg a.i./L test group a slight inhibition was observed after 72 hours. All other test groups showed similar or higher growth rates when compared to the control. There was no dose response relationship observed. Due to the lack of inhibitory effects, no NOEC was defined. Hence, based on the obtained data, the 72 -hour and 96-hour ErC50 is > 960 mg a.i./L.

This study is considered to be reliable with restrictions. The cell concentration increase was slightly below the validity criterium. However, the results were close to the trigger and hence the results may be regarded together with further information as relevant for the risk assessment.