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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The rationale for the read-across approach is attached in section 13. See the link in the 'Cross-reference' table.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.53 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.881-8.51 mg/L / based on read-across
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.23 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on read-across
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 April 2006 - 19 May 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted 7 June 1984
Deviations:
yes
Remarks:
In the controls and the 2 lowest test concentrations the pH increased by >1 unit during the course of the test. An explanation for this deviation was not provided.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, and all test concentrations
- Sampling method: At 0 hours, samples (20 mL) of each test media were taken for chemical analysis from the test media preparation flasks. At 72 hours, inoculated replicate test vessels at each treatment level were pooled and samples of each were taken (20 mL) for chemical analysis.
- Sample storage conditions before analysis: not reported.
Vehicle:
no
Remarks:
Trials were performed to check if a solvent vehicle would improve solubility of the substance in test media. It was concluded that there was no advantage of using a solvent, and therefore test media were prepared by direct addition of the substance.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Each test media was prepared individually by direct addition of the test substance (0.411, 1.23, 3.7, 11.1, 33.3 and 100.04 mg) to a 1 litre volumetric flask and made up to volume with alga media (according to EC C.3, but with slightly higher MgCl2*6H20 concnetration). The contents of each flask were treated with 30 minutes ultrasound and stirred overnight in the dark. Following stirring, each test media was filtered through a 0.45 μm pore size filter.
- Controls: The control treatment was prepared by adding EC medium only to control test vessels.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Oban, UK (On receipt from the CCAP, the slope culture was stored in the fridge at 1-10°C. A typical shelf life for each slope was 8 months.)
- Age of inoculum (at test initiation): approx. 72 h
- Method of cultivation: Prior to each test, a sub-sample (ca 200 μL) of a current concentrated liquid slope culture was added to two starter cultures (conical flasks) containing 100 mL of alga nutrient media (EC). Each culture was incubated (under the same conditions as used in the test) for approximately 72 hours prior to the start of each test. The second (unused) starter culture (incubated as a contingency) was discarded after the test was started.

ACCLIMATION
- Acclimation period: none reported
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24.3-24.6°C
pH:
t=0: 7.9-8.3 (measured in bulk flasks, with and without algal cells)
t=72: 8.0 (measured in individual vessles without algal cells)
t=72: 8.0-10.2 (measured in individual vessels with algal cells)
Nominal and measured concentrations:
- Nominal: 0.411, 1.23, 3.70, 11.1, 33.3 and 100 mg/L (direct addition of the test substance to algal medium)
- Measured: 0.313, 1.00, 2.85, 8.43, 14.4 and 8.83 mg/L (mean values of 0-hour (without algae) and 72-hour (with algae))
- Recovery: 76, 81, 77, 76, 43 and 8.8 % of the nominal concentrations
Details on test conditions:
TEST SYSTEM
- Test vessel: sterile (autoclaved) 250 mL Erlenmeyer (conical) flasks, loosely-capped
- Fill volume: 100 mL
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 178 x 10^4 cells/mL (mean, n=6)
- No. of vessels per concentration (replicates): 3 (+1 vessel/concentration without algae to establish background counts on each sampling occasion)
- No. of vessels per control (replicates): 6 (+1 vessel without algae to establish background counts on each sampling occasion)

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD 201; stock solutions containing the various nutrients were prepared with reverse osmosis (RO) water. Aliquots of each of the stock solutions were then added to RO water. The alga culture medium was autoclaved. Once cooled, an aliquot of a stock solution of NaHCO3 was added to the alga culture medium through a sterile filter (0.2 μm filter pore size).
- Culture medium different from test medium: no
- Intervals of water quality measurement:
* At the start of the test, the pH of freshly prepared test media, before and after alga cell inoculation was determined.
* The incubator temperature (minimum and maximum) was recorded continuously using a digital temperature logger with the probe maintained in the incubator in an additional vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: t=0: 7060 lux, t=72 h: 7200 lux (cooled white fluorescent tube lights, emitting white light across the visible portion of the spectrum (400 - 700 nm))

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: particle counter (C2 Coulter Counter)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 3
- Range finding study: yes
- Range finding test concentrations: 0.1, 1, 10 and 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.53 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.881-8.51 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.23 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none specifically reported
- Other: In the control and the two lowest test concentrations (with algal cells) the pH values were 10.0-10.2 after 72 hours. This is >2 pH units higher than the pH values measured at test start (7.9-8.0). An explanation for this finding has not been provided in the report. It should be noted that the corresponding treatment groups without algal cells did not show increased pH.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: no

Table: Growth of algae cultures exposed to test material during the definitive test

Nominal concentration

Cell concentration

(cells x 10000/mL)

Mean area under growth curve

(A x 1000000)

Average specific growth rate

(µ x 0.01)

24-h

48-h

72-h

0-24-h

0-48-h

0-72-h

0-24-h

0-48-h

0-72-h

control

7.34

41.1

197

 

 

 

 

 

 

6.30

34.1

169

6.77

40.0

203

6.82

36.7

165

6.77

36.9

181

6.00

32.2

156

Mean

6.67

36.833

179

0.68

5.66

31.2

7.9

7.505

7.194

0.41

6.10

39.2

197

 

 

 

 

 

 

6.13

35.4

178

7.46

42.2

174

Mean

6.56

39.0

183

0.667

5.89

32.3

7.82

7.625

7.233

1.23

7.00

39.3

197

 

 

 

 

 

 

6.03

31.1

162

5.88

35.1

166

Mean

6.30

35.2

175

0.636

5.37

30.3

7.66

7.407

7.166

3.70

3.67

13.0

58.7

 

 

 

 

 

 

3.78

9.47

21.1

5.30

16.4

49.9

Mean

4.25

12.9

43.2

0.39

2.21

8.72

5.97

5.283

5.108

11.10

2.39

4.90

12.0

 

 

 

 

 

 

2.31

5.13

5.66

2.15

3.9

7.28

Mean

2.28

4.63

8.32

0.154

0.743

2.06

3.43

3.178

2.873

33.30

0.80

0.802

0.122

 

 

 

 

 

 

1.08

0.666

0.246

1.14

1.08

0.064

Mean

1.00

0.849

0.144

0.00032

-0.0174

-0.138

-0.037

-0.382

-2.896

100.00

0.93

-0.006

0.012

 

 

 

 

 

 

0.89

-0.040

0.002

1.14

0.078

0.042

Mean

0.99

0.011

0.019

-0.00132

-0.121

-0.358

-0.071

0.000

-6.392

 

Table: Inhibition (%) in growth of algae exposed to test material relative to the Control treatment

Nominal concentration

Reduction in average area under the

growth curve relative to the control (%)

Reduction in average specific growth

rates relative to the control (%)

0-24-h

0-48-h

0-72-h

0-24-h

0-48-h

0-72-h

Control

NA

NA

NA

NA

NA

NA

0.411

1.85

-4.11

-3.28

0.96

-1.60

-0.540

1.23

6.43

5.06

2.97

3.01

1.31

0.390

3.70

42.7

60.9

72.1

24.4

29.6

29.0

11.1

77.4

86.9

93.4

56.5

57.7

60.1

33.3

100

100

100

100

105

140

100

100

102

101

101

100

189

TEST MEDIA DESCRIPTIONS

- At the start of the exposure period, the control and test media at all nominal concentrations appeared as clear, colourless solutions.

- At the end of the exposure period (72 hours), the control and test media at all nominal concentrations not inoculated with algae appeared as clear, colourless solutions.

- At the end of the exposure period (72 hours), the control and test media at all nominal concentrations inoculated with algae appeared as green to colourless (low to high concentration) with a hazy dispersion of algal cells.

Validity criteria fulfilled:
yes
Remarks:
see 'Overall remarks'.
Conclusions:
The 72-h ErC50 and 72-h NOErC values are 4.53 mg/L, and 1.23 mg/L, respectively, in freshwater algae (P. subcapitata).
Executive summary:

The toxicity to freshwater algae (P. subcapitata) was examined in a study according to OECD TG 201, and in compliance with GLP criteria. Fresh cultures of algae at a starting cell concentration of 1E+04 cells/mL, were exposed to nominal test concentrations of 0 (control), 0.411, 1.23, 3.70, 11.1, 33.3 and 100 mg/L for 72 hours. Test substance analysis was performed and arithmetic mean measured concentrations determined to be 0.313, 1.00, 2.85, 8.43, 14.4 and 8.83 mg/L (mean values of analysis at 0-hour (without algae) and 72-hour (with algae)). As measured concentrations were not within ±20% of nominal, effect values are expressed as measured concentrations. In the control and the two lowest test concentrations (with algal cells) the pH values were >2 pH units higher than the pH values measured at test start. An explanation for this finding has not been provided in the report. It should be noted that the corresponding treatment groups without algal cells did not show increased pH. As, however, increase in cell density in controls was at least a factor 16, this is not considered to have significantly affected the outcome of the test. Algal cell concentrations were measured at 24, 48, and 72 hours after the start of the test, and inhibition of algal specific growth rates calculated.

A concentration dependent reduction of growth rate was observed throughout the test. The 72 -h ErC50 and 72 -h NOErC values were determined to be 4.53 mg/L and 1.23 mg/L, respectively.

Description of key information

The acute toxicity of the substance to freshwater algae (P. subcapitata) was derived by read-across from an analogue with the same chemical core structure, which was examined for algae toxicity in a study performed in accordance with OECD TG 201, and in compliance with GLP criteria.

Fresh cultures of algae at a starting cell concentration of 1E+04 cells/mL, were exposed to nominal test concentrations of 0 (control), 0.411, 1.23, 3.70, 11.1, 33.3 and 100 mg/L for 72 hours.

Test substance analysis was performed at the start (t = 0 h) and at the end of exposure (t = 72 h), by means of a sufficiently validated HPLC-UV method. Since measured concentrations were not within ±20% of nominal, results are expressed as arithmetic mean measured concentrations, which were determined as 0.313, 1.00, 2.85, 8.43, 14.4 and 8.83 mg/L (mean values of analysis at 0-hour (without algae) and 72-hour (with algae)).

In the control and the two lowest test concentrations (with algal cells) the pH values were >2 pH units higher than the pH values measured at test start. An explanation for this finding has not been provided in the report. It should be noted that the corresponding treatment groups without algal cells did not show increased pH. As, however, increase in cell density in controls was at least a factor 16, this is not considered to have significantly affected the outcome of the test. Algal cell concentrations were measured at 24, 48, and 72 hours after the start of the test, and inhibition of algal specific growth rates calculated.

A concentration dependent reduction of growth rate was observed throughout the test. The 72 -h ErC50 and 72 -h NOErC values were determined to be 4.53 mg/L and 1.23 mg/L, respectively.

All study validity criteria were met.

Key value for chemical safety assessment

EC50 for freshwater algae:
4.53 mg/L
EC10 or NOEC for freshwater algae:
1.23 mg/L

Additional information