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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:
72 hours
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals. Test Guideline 201, Freshwater Alga and Cyanobacteria, Growth Inhibition Test. Adopted 23 March 2006
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test substance name Crodateric CYAP
Scymaris reference number 1014TS047
Physical state and appearance Dark solid
Supplier Croda Europe Ltd.
Lot/batch number PS-196-555
Analytical monitoring:
yes
Vehicle:
yes
Remarks:
The culture medium used for the test, and for the maintenance of cultures of the alga used as inoculum for the test was AAP-medium
Details on test solutions:
This study was run with a culture medium control and nominal exposure concentrations of 0.19, 0.609, 1.95, 6.25 and 20 mg/L.
Control and mean measured concentrations of 0.18, 0.57, 1.8, 5.8 and 18 mg/L
A primary stock concentrate of Crodateric CYAP with a nominal concentration of 20 mg/L was prepared by weighing a nominal 0.02 g of test substance (actual weight: 0.01992 g) and making up to 1000 mL volume with the culture medium AAP in a volumetric flask. The stock was stirred for 40 minutes. The resultant stock was observed to be a clear and colourless solution and was used to prepare the test solutions. This was achieved by adding the relevant volumes of the primary stock to AAP media and making up to 1000 mL volume in a volumetric flask. The 20 mg/L stock was used directly for the 20 mg/L blank and test vessels. The control consisted of culture medium only.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test species was the unicellular green alga Pseudokirchneriella subcapitata strain CCAP 278/4 from laboratory cultures maintained under axenic conditions. A 3-day old culture of the alga in the exponential growth phase was used as inoculum for the test. The culture was grown in the medium, and under the environmental conditions, described for the test.
Test type:
static
Water media type:
other: Cultre medium
Total exposure duration:
72 h
Test temperature:
22 +/- 2C
pH:
At the start of the test the pH ranged from 7.51 and 8.02 and at the end of the test the range was from 7.67 to 7.76
Details on test conditions:
The test vessels were glass conical flasks of 250 mL nominal capacity closed with foam bungs. Each flask contained 100 mL of test solution. The cultures were incubated at 22 +/- 2°C (the nominal test temperature), under continuous "cool-white" illumination of approximately 6000 lux, with nominal orbital shaking at 160 rpm.
Six replicates of the culture medium control and triplicates of each concentration of the test substance were employed. The position of each test replicate vessel in the incubator was randomised daily. One blank vessel (without algal inoculum) was incubated concurrently for each control and test concentration sampling occasion.
The algal cell densities of the inoculum and test cultures were determined by electronic particle counting, using a Coulter counter and counting between a lower and upper threshold equivalent spherical diameter of approximately 2.3 and 5.0 µm respectively. Each replicate test vessel was inoculated with 0.514 mL of the inoculum culture to give a nominal cell density of 0.5 × 104 cells/mL. One 100 mL volume of Coulter electrolyte, inoculated in the same manner, had a cell density of 0.539 × 104 cells/mL. This value was used for growth calculations.
After 24, 48, and 72 hours, samples were removed from each test and blank vessel. The appropriate blank particle count was subtracted from that of the test culture to obtain the cell density.
At the end of the test microscopic observations were made on samples taken from a single replicate of the control and each test substance concentration.
Reference substance (positive control):
yes
Remarks:
culture medium
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.25 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
20 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Key result
Duration:
72 h
Dose descriptor:
other: ErC50
Effect conc.:
> 20 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
other: EyC50
Effect conc.:
13.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
The microscopic observations, made at the end of the test, showed that compared to the control the algal cells sampled from the nominal 0.19, 0.609, 1.95, 6.25 mg/L test concentrations appeared normal and the cells sampled from the nominal 20 mg/L test concentration appeared deformed

Growth rates - The growth rate (0 to 72 hours) was calculated for each replicate culture (Table 3), according to the formula:

where N1       =         Cell density at start of test

           N2       =         Celldensity at end of test

           t          =         Time interval (days)

The control was compared to the treatments by one-way analysis of variance and Bonferroni adjusted t test to identify significant differences (p<0.05). ECXwas calculated using the linear interpolation (ICPIN) method.

The mean growth rates, together with the rates expressed as percentages of the control.

The results obtained from these growth rate analyses, based on mean measured test concentrations, were as follows:

 

Crodateric CYAP

(mg/L)

95% confidence limits

(mg/L)

NOEC

6.25

-

LOEC

20

-

ErC50

>20

N/A

ErC20

13.7

11.7-15.3

ErC10

8.90

6.58-10.7

 

Yield - This response is defined as the biomass at the end of the test minus the starting biomass. For the purposes of calculation, the cell particle density count (cell particles per unit volume) is an acceptable surrogate for biomass. The control was compared to the treatments by one-way analysis of variance and Bonferroni adjusted t test to identify significant differences (p<0.05). ECXwas calculated using the linear interpolation (ICPIN) method.

The EC50, EC20and EC10values with their associated confidence intervals were subsequently calculated using the Linear Interpolation method. The yield mean values are shown inTable 6, together with the yields expressed as percentages of the control.

The results obtained from these statistical analysis, based on mean measured test concentrations, were as follows:

 

 

Crodateric CYAP

(mg/L)

95% confidence limits

(mg/L)

NOEC

6.25

-

LOEC

20

-

EyC50

13.7

11.1-15.5

EyC20

6.74

N/A

EyC10

1.93

N/A

Validity criteria fulfilled:
yes
Conclusions:
The microscopic observations, made at the end of the test, showed that compared to the control the algal cells sampled from the nominal 0.19, 0.609, 1.95, 6.25 mg/L test concentrations appeared normal and the cells sampled from the nominal 20 mg/L test concentration appeared deformed.
The NOEC level for both growth rate and yield for this substance was determined to be 6.25 mg/l and the LOEC level for both growth rate and yield was determined to be 20 mg/l

Description of key information

The microscopic observations, made at the end of the test, showed that compared to the control the algal cells sampled from the nominal 0.19, 0.609, 1.95, 6.25 mg/L test concentrations appeared normal and the cells sampled from the nominal 20 mg/L test concentration appeared deformed.

The NOEC level for both growth rate and yield for this substance was determined to be 6.25 mg/l and the LOEC level for both growth rate and yield was determined to be 20 mg/l.

ErC50 Growth rate = >20 mg/l. EyC50 Yield = 13.7 mg/l

Key value for chemical safety assessment

EC50 for freshwater algae:
20 mg/L
EC10 or NOEC for freshwater algae:
6.25 mg/L

Additional information