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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:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
None.
Analytical monitoring:
yes
Details on sampling:
The test substance concentration was determined in one replicate of each treatment at the beginning of the experiment and in all replicates at the end of the experiment.
Vehicle:
no
Details on test solutions:
30 mg of the test substance was weighed out and dissolved in 1L of test medium to obtain a 30 mg/L nominal test medium. This stock solution was filtered for sterilization. This stock solution was diluted to obtain a series a test concentrations.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The testing used Pseudokirchneriella subcapitata (Former name: Selenastrum capricornutum) which is an unicellular green alga. This species was strain ATCC22662 obtained from the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852 USA), and was aseptically cultured at this facility. The algae provided for the test used those cultured for 4 days prior to the start of the experiment under conditions identical to the test conditions. This time period allows for the cultured cells to enter the exponential growth period. No heteromorphic or abnormal cells were seen and so the experiment was started.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
None.
Post exposure observation period:
None.
Hardness:
Not measured.
Test temperature:
23°C
pH:
7.8 - 9.2 (min-max)
Dissolved oxygen:
Not measured
Salinity:
Not measured (freshwater)
Conductivity:
Not measured
Nominal and measured concentrations:
Nominal concentrations: 0 ; 1.3 ; 2.8 ; 6.2 ; 13.6 ; 30 mg/L (dilutions were made from a unique stock solution at 28.7 mg/L and a spacing factor of 1.8)
Mean measured concentrations: 0 ; 0.95 ; 2.02 ; 4.47 ; 9.69 ; 21.55 mg/L
Results were based on mean measured concentrations because measured concentrations were in the [80-120]% range of the nominal concentrations. However, it has to be noted that there were almost no variation (< 1%) between initial (0 hours) and final (72 hour) measurements.
Details on test conditions:
TEST SYSTEM
- Material: 300 mL glass Erlenmeyer flask filled with 100 mL test medium and stoppered with aerated silicone bung.
- Initial cells density: 10000 per mL medium
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

OTHER TEST CONDITIONS
- Sterile test conditions: yes, either by autoclaving or filtering
- Adjustment of pH: not necessary
- Photoperiod: LL (continuous lightning)
- Light intensity and quality: 4000 - 5000 lux

The number of pre-cultured algae cells was counted, and 1.382 mL of the pre-culture medium was added to each replicate (300 mL glass flask with 100 mL test medium) so that the cell concentration in the test medium was 10000 cells/mL. Each of the test flasks was set in the incubation device at 23.0±2.0°C and the experiment started. Thereafter, the cell concentration was measured with a particle count analysis device after 24, 48, and 72 hours.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
Data on measured concentrations are given in table 1. Cell biomass recorded per day and per replicate is given in table 2.
Results with reference substance (positive control):
Growth inhibition testing was performed on the algae using a standard substance (potassium dichromate, reagent grade) in January 2005. Test Value was 0.62 mg/L (Background Data: 0.63±0.23 mg/L). However, there is no indication in the report on what this value actually is (EC50, EC10, NOEC ?), on which endpoint it lies (biomass, growth rate inhibition ?) and how the results were statistically analysed.
Reported statistics and error estimates:
Results were re-analysed with Toxrat 3.2.1 (see attached Toxrat report) for growth rate inhibition. All three validity criteria passed (growth > 16 ; CVinter < 7% ; CVintra < 35%). The regression analysis was performed according to a 3 parameter-logistic regression based on Levenberg-Marquadt optimization method (1000 cycles). Confidence limits were estimated by bootstrapping (10 000 resamplings).

Table 1: Nominal and measured concentrations at 0 and 72 hour.

Nominal concentration (mg/L)

Measured concentrations (mg/L)

Arithmetic mean (mg/L)

0-hour

72-hour

Control

Not detected

Not detected

0

1.3

0.96

0.93

0.95

2.8

2.01

2.02

2.02

6.2

4.46

4.47

4.47

13.6

9.64

9.73

9.69

30

21.51

21.59

21.55

Table 2: algal concentrations at 24 hour (x10 000 cells/mL). Initial concentrations were 10 000 cells/mL

 

Mean measured concentrations

 

Control

0.95

2.02

4.47

9.69

21.55

Replicate 1

2.22

1.93

1.84

1.38

1.22

0.943

Replicate 2

2.42

2.04

1.75

1.05

1.28

1.11

Replicate 3

2.59

2.03

1.62

1.37

1.24

1.01

Replicate 4

2.56

 

 

 

 

 

Replicate 5

2.19

 

 

 

 

 

Replicate 6

2.34

 

 

 

 

 

Table 3: algal concentrations at 48 hour (x10 000 cells/mL). Initial concentrations were 10 000 cells/mL

 

Mean measured concentrations

 

Control

0.95

2.02

4.47

9.69

21.55

Replicate 1

11.7

10.2

7.19

5.12

3.61

2.52

Replicate 2

10.7

10.2

7.59

3.37

3.73

2.51

Replicate 3

10.4

10.5

7.1

4.69

3.56

2.56

Replicate 4

11.6

 

 

 

 

 

Replicate 5

11.6

 

 

 

 

 

Replicate 6

12.3

 

 

 

 

 

Table 4: algal concentrations at 72 hour (x10 000 cells/mL). Initial concentrations were 10 000 cells/mL

 

Mean measured concentrations

 

Control

0.95

2.02

4.47

9.69

21.55

Replicate 1

49.7

52.7

24.8

15.8

11.2

7.23

Replicate 2

46.4

50.6

26.9

10.8

11.3

6.97

Replicate 3

50.1

45.1

25.7

14.6

12.1

7.21

Replicate 4

52.2

 

 

 

 

 

Replicate 5

52.4

 

 

 

 

 

Replicate 6

51.0

 

 

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
A GLP test following a protocol equivalent to OECD 201 guideline was conducted on Pseudokirchneriella subcapitata. Mean measured concentrations were 0 ; 0.95 ; 2.02 ; 4.47 ; 6.69 ; 21.55 mg/L. Growth rate inhibition EC50 and EC10 were 17.9 and 0.8 mg/L respectively.
Executive summary:

A GLP test following a protocol equivalent to OECD 201 guideline was conducted on Pseudokirchneriella subcapitata. Mean measured tested concentrations were 0 ; 0.95 ; 2.02 ; 4.47 ; 6.69 ; 21.55 mg/L. All treated groups were tested in triplicate and the control group was performed in hexaplicate. Test was initiated at 10 000 cells/mL and conducted at 23°C under constant lightning around 4000 -5000 lux. Cell concentration was measured every 24 hours. The test was completed after 72 hours. All validity criteria passed (growth > 16 ; CVinter < 7% ; CVintra < 35%). Growth rate inhibition EC50 and EC10 were 17.9 and 0.8 mg/L respectively.

Description of key information

A GLP test following a protocol equivalent to OECD 201 guideline was conducted on Pseudokirchneriella subcapitata. Mean measured tested concentrations were 0 ; 0.95 ; 2.02 ; 4.47 ; 6.69 ; 21.55 mg/L. All treated groups were tested in triplicate and the control group was performed in hexaplicate. Test was initiated at 10 000 cells/mL and conducted at 23°C under constant lightning around 4000 -5000 lux. Cell concentration was measured every 24 hours. The test was completed after 72 hours. All validity criteria passed (growth > 16 ; CVinter < 7% ; CVintra < 35%). Growth rate inhibition EC50 and EC10 were 17.9 and 0.8 mg/L respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
17.9 mg/L
EC10 or NOEC for freshwater algae:
0.8 mg/L

Additional information