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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
not specified
Analytical monitoring:
no
Details on sampling:
Samples were taken at 0, 24, 48 and 72 hours and the absorbance measured at 665 nm using a Jenway 6100 spectrophotometer.
Vehicle:
no
Details on test solutions:
Test concentrations - 3.125, 6.25, 12.5, 25 and 50 mg/L

Culture medium
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 0.08 mg/L
Na2EDTA.2H2O 0.1 mg/L
H3BO3 0.185 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 3x10-3 mg/L
CoCl2.6H2O 1.5x10-3 mg/L
CuCl2.2H2O 1x10-5 mg/L
Na2MoO4.2H2O 7x10-3 mg/L
NaHCO3 50 mg/L
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 276/20
Test type:
static
Water media type:
not specified
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No data
Hardness:
No data
Test temperature:
24 degrees centigrade
pH:
The pH of the culture medium after equilibration with air was approximately 8
Dissolved oxygen:
No data
Salinity:
No data

Nominal and measured concentrations:
Test concentrations: 3.125, 6.25, 12.5, 25 and 50 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL
- Type: closed and loosely stoppered to reduce evaporation
- Control end cells density: 1.55 x 10 E+04
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity and quality: Approximately 7000 lux
- shaking culture (100 rpm)

EFFECT PARAMETERS MEASURED: Growth inhibition
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
14 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: No statistically significant differences were found between control, 3.125 and 6.25 mg/L concentrations whilst all other test concentrations were significantly different
Details on results:
From the data given in Table 1 (below) and presented graphically in Figure 1 (attached) it can be seen that the absorbance values were sufficiently well correlated with cell density values and therefore it was considered justifiable to monitor the growth of the test cultures by absorbance values alone.

From the data given in Table 2 and Table 3 (below), it is clear that both the growth (r) and the biomass (b) of Scenedesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72 hour exposure period.

Accordingly the following results were determined from the data:

EbC50 (72h): 12 mg/L
ErC50 (0-24h): 14 mg/L

Where EbC50 is the test concentration that reduced biomass by 50 % and and ErC50 is the test concentration that reduced specific growth rate by 50 %.

There were no statistically significant differences between the control, 3.125 and 6.25 mg/L concentrations (P ≥ 0.05%) however all other test concentrations were significantly different (P < 0.001) and, therefore, the No Observed Effect Concentration (NOEC) is given as 6.25 mg/L.

The following data show that the cell concentration of the control cultures increased by a factor of 17 during the test in line with the OECD guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

There were no abnormalities detected in any of the control or test cultures during microscopic examination at 72 hours.
Results with reference substance (positive control):
No data
Reported statistics and error estimates:
One way analysis of variance was carried out on the area under the growth curve data at 72 hours for the control and all test concentrations to determine any statistically significant differences between the test control and control groups.

Table 1 - Mean cell density and absorbance values of the control cultures

 

0h

24h

48h

72h

Mean cell density* (cells/mL)

1.55 x 104

7.72 x 104

1.76 x 105

2.62 x 105

Absorbance

0.026

0.086

0.196

0.397

* Mean cell density values represent the mean number of cells per mL calculated from the mean of the cell counts from three fields of view for each of the replicate flasks

Table 2 - Absorbance and pH values

Nominal Concentration (mg/L)

pH

Absorbance values

pH

0 h

24 h

48 h

72 h

72 h

Control

R1

8.0

0.026

0.088

0.392

10.0

 

R2

8.0

0.026

0.082

0.398

10.0

 

R3

8.0

0.026

0.087

0.401

10.0

 

x

 

0.026

0.086

0.397

 

3.125

R1

8.0

0.026

0.090

0.0392

10.0

 

R2

8.0

0.026

0.086

0.393

10.0

 

R3

8.0

0.026

0.088

0.395

10.0

 

x

 

0.026

0.088

0.393

 

6.25

R1

8.0

0.026

0.087

0.401

10.0

 

R2

8.0

0.026

0.089

0.388

10.0

 

R3

8.0

0.026

0.090

0.389

10.1

 

x

 

0.026

0.089

0.393

 

12.5

R1

8.0

0.026

0.050

0.188

10.0

 

R2

8.0

0.026

0.046

0.192

10.0

 

R3

8.0

0.026

0.047

0.199

9.9

 

x

 

0.026

0.048

0.193

 

25

R1

8.0

0.026

0.030

0.030

9.9

 

R2

8.0

0.026

0.032

0.032

10.0

 

R3

8.0

0.026

0.031

0.031

9.9

 

x

 

0.026

0.031

0.031

 

50

R1

8.0

0.026

0.026

0.026

9.9

 

R2

8.0

0.026

0.027

0.026

9.9

 

R3

8.0

0.026

0.026

0.026

9.9

 

x

 

0.026

0.026

0.026

 

R1, R2, R3 = Replicates 1,2 and 3

= Mean value

Table 3 - Inhibition of growth

Nominal Concentration (mg/L)

Area under curve at 72 h

% Inhibition

Growth rate (0-24 h)

% Inhibition

Control

9.956

-

0.050

-

3.125

9.962

0

0.051

[2]

6.25

9.992

0

0.051

[2]

12.5

4.052

59

0.026

48

25

0.300

97

0.007

86

50

0.008

100

0.000

100

EbC50 (72h) from Figure 3 = 12 mg/L

ErC50 (0-24) from Figure 3 = 14 mg/L

Validity criteria fulfilled:
yes
Conclusions:
The EC50 value (72h) was 12 mg/L based on biomass. The corresponding value (0-24h) based on growth rate was 14 mg/L. The NOEC value was considered to be 6.25 mg/L based on statistical differences.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
concentration of test material after 72 hours was found to decrease with decreased initial test material concentration. The effect was thought to be due transfer of the test material into the algae but results were still based on nominal concentrations.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The concentration of the test material was measured at the start of the study and after 72 hours. The number of cells were counted at 24, 48 and 72 hours.
Vehicle:
no
Details on test solutions:
Nominal concentrations: Control, 1.00, 2.19, 4.78, 10.5, 22.9, 50.0 mg/L
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
No data
Test type:
static
Water media type:
not specified
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No data
Hardness:
No data
Test temperature:
23 ± 2 degrees Centrigrade.
pH:
pH 7.8 - 10.2
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Nominal concentrations: Control, 1.00, 2.19, 4.78, 10.5, 22.9, 50.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL
- Initial cells density: 1 x 10 E+04
- No. of vessels per concentration (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes - see Table A-1 (attached)

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity and quality: 4000 lux (within ± 20 % variation near liquid surface)
- Shaking culture (100 rpm)

EFFECT PARAMETERS MEASURED: Growth inhibition
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% confidence limits could not be calculated
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
32.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits could not be calculated
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
33.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits could not be calculated
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
10.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
22.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
There was no measured concentration exceeding ± 20 % of the nominal concentration at the start, therefore, the nominal concentrations were used for the calculation of the growth-inhibition concentration. Representative chromatograms are attached.

The concentrations of the test material after 72 hr-exposure were 0.65 ~ 48.2 mg/L, and their ratios to the nominal values were 65~95 %. As the lower concentration groups showed the larger decrease of the concentration, the main cause of the decrease of the concentrations was considered to be transfer of the test material into algae.

The cell density in the control group increased up to 196 times in mean value after 72-hr culture, which showed the normal growth under the test condition. The cell density in each concentration group showed a tendency to decrease as the material concentration increased (dose-dependently).

The following results were obtained:

Inhibition concentration by the comparison of the area under the growth curve:
EbC50 (0-72): 20.3 mg/L (95% confidence division: unable to be calculated)
NOECb (0-72): 10.5 mg/L.

Inhibition concentration by the comparison of growth rate:
ErC50 (24-48) : 32.1 mg/L (95% confidence division: unable to be calculated).
NOECr (24-48): 10.5 mg/L
ErC50 (24-72) : 33.3 mg/L (95% confidence division: unable to be calculated)
NOECr (24-72): 22.9 mg/L

The temperatures were within the nominal range (23 ± 2 degrees Centigrade). The pH values of the test solution were 7.8 ~ 9.3 at the start of exposure. The pH values shown in the groups of the higher concentration were slightly high. This is considered to be due to the slight alkaline nature of the test material. The pH values at the end of the study were 8.4 ~ 10.2. When carbonic acid assimilation (photosynthesis) was vigorous and the growth rate of algae was high, pH may be sometimes elevated 1 or more. In this study, the pH values in the control group and the groups of 2.19 mg/L or lower increased 1 or more in comparison with the initiation values.
Results with reference substance (positive control):
No data
Reported statistics and error estimates:
The EC50 and associated 95 % confidence limits were determined by least squares linear regression analysis of the logarithm of nominal test concentration against percent growth inhibition relative to the control. Table 4 (attached) states the concentrations used in regression analyses.

The NOEC values were determined by an analysis of variance (ANOVA), Dunnett test, subsequent to Bartlett test for homogeneity of variances. Statistical analyses were performed using Yukms Statlight #4 software (Yukms Corp, Tokyo) and all tests of significance were at α = 0.05, except the Bartlett test, which was at α = 0.01.

The concentration of the test material in the test solution was measured at the start of the study and after 72 hours. Results are shown in Table 1.

Table 1 - Measured concentrations of the Test Substance in Test Water

Nominal Concentration (mg/L)

Measured Concentration (mg/L)

0 Hour

Percent of Nominal

72 Hour

Percent of Nominal

Control

<0.02

--

<0.02

--

1.00

0.98

98

0.65

65

2.19

2.14

98

1.53

70

4.78

4.67

98

4.01

84

10.5

10.2

97

9.37

89

22.9

22.3

97

21.7

95

50.0

48.4

97

48.2

96

Cell densities during the period of exposure are shown in Table 2 (attached), and the growth curve is shown in Figure 1 (attached).

The growth-inhibition ratio in each concentration group is shown in Table 3 (attached), and 50 % growth inhibition concentration (EC50) and maximum No Observed Effect Concentration (NOEC) are shown in Table 4 (below). The concentration-inhibition ratio curves are shown in Figures 2 and 3 (attached).

Table 4 - Calculated EC50 and NOEC

Based on IA(0-72h) value (Areas under growth curve)

EbC50 (0-72) (mg/L)

95-Percent Confidence Limits (mg/L)

NOECb (0-72) (mg/L)

20.3*1

--

10.5

Based on Im (24-48h) value (Growth rates)

ErC50 (24-48) (mg/L)

95-Percent Confidence Limits (mg/L)

NOECr (24-48) (mg/L)

32.1*2

--

10.5

Based on Im (24-72) value (Growth rates)

ErC50 (24-72) (mg/L)

95-Percent Confidence Limits (mg/L)

NOECr (24-72) (mg/L)

33.3*2

--

22.9

*1 using the concentrations of 10.5 and 22.9 mg/L in the regression analysis

*2 using the concentrations of 22.9 and 50.0 mg/L in the regression analysis

-- not calculated

The temperatures in the incubation chamber during the exposure period of 72 hours are shown in Table 5 (attached), and pH value of test solution is shown in Table 6 (attached).

Validity criteria fulfilled:
yes
Conclusions:
An EC50 value was calculated as 20.3 mg/L (0-72h) based on biomass. The corresponding EC50 values were 32.1 mg/L (24-48h) and 33.3 mg/L (24-72h) based on growth rate. The NOEC based on biomass (72 h) was reported as 10.5 mg/L. The corresponding NOECs based on growth rate were 10.5 mg/L (24 h) and 22.9 mg/L (72h).

The NOEC based on biomass was calculated as 10.5 mg/L (0-72h). Corresponding NOEC values based on growth rate were 10.5 mg/L (24-48h) and 22.9 mg/L (24-72h).

Description of key information

EC50 (72 h) 20.3 mg/L for Selenastrum capricornutum (OECD 201)

NOEC (72 h) 10.5 mg/L for Selenastrum capricornutum (OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
20.3 mg/L
EC10 or NOEC for freshwater algae:
10.5 mg/L

Additional information

Two studies are available to describe the long-term effect of test material on algae. The most reliable of those studies is that conducted by the Japanese government (1996) to GLP standards and with analytical monitoring and this has been selected as the key study.

A supporting study (Mead, 1995) fails to provide an EC50 (72 h) based on growth rate and derives the NOEC via a less rigorous method than the key study.  According to ECHA guidance on information requirements and chemical safety assessment Chapter R.10: Characterisation of dose [concentration]-response for environment, two long-term results (such as EC10 or NOECs) from species representing two trophic levels (fish and/or Daphnia and/or algae) permit an assessment factor of 50 to be applied when calculating the freshwater PNEC.  

The availability of a GLP study with analytical monitoring (Ministry of Environment, Japan, 1996), describing algal inhibition allows that data to be used in conjunction with long-term toxicity data from Daphnia magna (Ministry of Environment, Japan, 2000) during PNEC calculation.