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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:
20 April to 29 May 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline 201. All validity criteria were fulfilled
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
- Purity test date: 04 November 2014
- test substance storage: protected from light
- Vapour pressure: 1.51 Pa (calculated by sponsor); 5.8 Pa at 20°C (determined by WIL Research Europe); 11 Pa at 25°C (determined by WIL Research Europe)
- Solubility in water: 0.667 g/L at 20°C (determined at WIL Research Europe B.V.)
- Stability in water: Stable at pH 7
Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 h, t=24 h and t=72 h
Volume: 3.0 mL
Storage: Samples were stored in a freezer until analysis.
At the end of the exposure period, samples were taken from replicate 1 of all test solutions. In addition, samples were taken from replicate 2 at 20 mg/L, as this replicate contained a higher cell density compared to replicates 1 and 3 at this concentration.
Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at an intermediate substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 3.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L applying 15 minutes of magnetic stirring. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.
After preparation, volumes of 120 mL were added to each replicate of the respective test concentration. Subsequently, 2.4 mL of an algal suspension was added to each replicate providing a cell number of 10^4 cells/mL.
- Controls: Test medium without test substance or other additives.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24 °C.
- Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Pre-culture: 3-4 days before the start of the range-finding test and 4 days before the start of the final test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
22-23 °C
pH:
7.4-7.5 (0 h); 7.3-7.6 (0 h)
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
0.032, 0.16, 0.8, 4.0, 20 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 120 mL, airtight closed with no headspace
- Type: closed
- Initial cells density: An initial cell density of 1 x 10^4 cells/mL.
- Replicates: 3 replicates of each test concentration; 6 replicates of the control; 2 replicates at 4.0 mg/L without algae; 1 additional replicate of each test concentration with algae for sampling purposes at 24 hours

TEST MEDIUM / WATER PARAMETERS
- Adjusted M2 for volatile substances

OTHER TEST CONDITIONS
- Sterile test conditions: Yes.
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 80 to 81 μE.m2/s.
- Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank.
- Measurements
pH: At the beginning and at the end of the test.
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells: At the end of the final test microscopic observations were performed on the test concentration closest to the EC50 to observe for any abnormal appearance of the algae.

RANGE FINDING STUDY
First combined limit/range-finding test: Six replicates of exponentially growing algae were exposed to a control and a concentration of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
- Three replicates per concentration were exposed to 0.10, 1.0 and 10 mg/L.
- One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.
- An additional replicate without algae was included for the highest concentration for sampling purposes.
- pH was only measured in the control and the highest test concentration.
- Results used to determine the conditions for the definitive study: The expected EC50 for growth rate inhibition was between 10 and 100 mg/L. The expected EC50 for yield inhibition was close to 10 mg/L. Based on the results of the range-finding test, the dose levels, 0.032, 0.16, 0.8, 4.0, 20 and 100 mg/L have benn selected for definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L) - Project 509666
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
13 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval of 8.9 to 17 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
55 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval of 47 to 63 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
3.8 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Remarks on result:
other: 95% confidence interval of 1.1 to 6.6 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
17 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Remarks on result:
other: 95% confidence interval of 12 to 25 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Details on results:
Inhibition of growth rate and inhibition of yield:
- Growth rates were in the range of the controls at the TWA concentrations up to a TWA concentration of 3.2 mg/L during the 72-hour test period, whereas the growth rates of algae exposed to a TWA concentration of 16 and 82 mg/L were increasingly reduced. Statistically significant inhibition of growth rate was found at a TWA concentration of 16 and 82 mg/L.
- Inhibition of yield increased with increasing concentration of ST 05 C 14 from a TWA concentration of 3.2 mg/L upwards resulting in 96% inhibition at 82 mg/L. Statistically significant inhibition of yield was found at a TWA concentration of 16 and 82 mg/L.
- Microscopic observations at the end of the test at a TWA concentration of 16 mg/L revealed a normal and healthy appearance of the exposed cells when compared to the control.
Results with reference substance (positive control):
The EC50 for growth rate inhibition (72h-ERC50) was 1.3 mg/L with a 95% confidence interval ranging from 1.2 to 1.5 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (72h-EYC50) was 0.46 mg/L with a 95% confidence interval ranging from 0.43 to 0.49 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested was within the low end of this range.
Reported statistics and error estimates:
Determination of the NOEC and calculation of the EC50 : For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test, α=0.05, one-sided, smaller).
Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test substance.
The calculations were performed with ToxRat Professional v. 2.10.05 (ToxRat Solutions® GmbH, Germany).

Table 6.1.5/1: Percentage inhibition of growth rate and yield

 

Nominal concentration (mg/L)

TWA concentration (mg/L)

Mean

SD

n

% Inhibition

Percentage inhibition of growth rate

Control

-

1.509

0.0488

6

 -

0.03

-

1.505

0.0220

3

0.3

0.16

-

1.530

0.0384

3

-1.4

0.8

0.56

1.493

0.0142

3

1.1

4.0

3.2

1.477

0.0532

3

2.1

20

16

1.310

0.0934

3

13.2*

100

82

0.537

0.0970

3

64.4*

Percentage inhibition of yield

Control

-

92.467

14.6015

6

-

0.03

-

90.554

5.9511

3

 2.1

0.16

-

98.040

11.6292

3

-6

0.8

0.56

87.252

3.7318

3

5.6

4.0

3.2

83.804

14.0864

3

9.4

20

16

51.366

15.5694

3

44.4*

100

82

4.153

1.4436

3

95.5*

 

Measured test substance concentrations

At the start of the test, the actual test concentrations were in agreement with nominal except at the lowest concentration, for which the actual concentration represented 155% of nominal. At the end of the test, actual concentrations at 0.032 and 0.16 mg/L were below the limit of detection. At 0.8 mg/L the concentration decreased to 55% of initial. At 4.0, 20 and 100 mg/L the concentrations remained stable (80-100% of initial), except for the sample taken from vessel 2 of 20 mg/L (52% of initial). For 20 mg/L at the end of the exposure a weighted average concentration was calculated. Since, all concentrations, except at 100 mg/L, deviated from the nominal at the end of the test the Time weight Average concentrations were calculated to correspond with 0.52, 3.2, 16 and 82 mg/L for a concentration of 0.8, 4.0, 20 and 100 mg/L, respectively. At 0.032 and 0.16 mg/L no TWA concentration could be determined; these concentrations were not needed to determine the EC50 values. 

 

Experimental conditions:The pH was within the limits prescribed by the protocol (6.0-9.0, preferably not varying by more than 1.5 unit). During the exposure period the temperature measured in the incubator was maintained between 22 and 23 °C. Temperature remained within the limits prescribed by the protocol (21-24 °C, constant within 2 °C).

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, test substance reduced growth rate and inhibited the yield of this fresh water algae species significantly at time weight average exposure concentrations of 16 and 82 mg/L.
The EC50 for growth rate inhibition (72 h-ERC50) was at a TWA concentration of 55 mg/L with a 95% confidence interval ranging from 47 to 63 mg/L. The EC10 for growth rate inhibition (72h-ERC10) was at a TWA concentration of 13 mg/L with a 95% confidence interval ranging from 8.9 to 17 mg/L.
The 72 h-NOEC for growth rate was at a TWA concentration of 3.2 mg/L. As the ratio NOEC/LOEC is higher than 3.2, the NOEC for algae test is considered non acceptable.
Executive summary:

One key study is available (WIL, 2015) to assess the influence of the registered substance on the growth of the freshwater green algae species Pseudokirchneriella subcapitata in a 72h static condition, according to OECD Guideline 201 with GLP statement.

The algae were exposed to test substance at concentrations of 0.032, 0.16, 0.8, 4.0, 20 and 100mg/L. The study was conducted under static conditions with an initial cell density of 104 cells/mL and the total exposure period was 72 hours. Three replicates were tested for each test substance concentration and six replicates for the control. The environmental conditions were within the acceptable limits. Samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and after 72 hours of exposure.

At the start of the test, the actual test concentrations were in agreement with nominal except at the lowest concentration, for which the actual concentration represented 155% of nominal. At the end of the test, actual concentrations at 0.032 and 0.16 mg/L were below the limit of detection. At 0.8 mg/L the concentration decreased to 55% of initial. At 4.0, 20 and 100 mg/L the concentrations remained stable (80-100% of initial), except for the sample taken from vessel 2 of 20 mg/L (52% of initial). For 20 mg/L at the end of the exposure a weighted average concentration was calculated. Since, all concentrations, except at 100 mg/L, deviated from the nominal at the end of the test the Time weight Average concentrations were calculated to correspond with 0.52, 3.2, 16 and 82 mg/L for a concentration of 0.8, 4.0, 20 and 100 mg/L, respectively. At 0.032 and 0.16 mg/L no TWA concentration could be determined; these concentrations were not needed to determine the EC50 values.

Growth rates were in the range of the controls at the TWA concentrations up to a TWA concentration of 3.2 mg/L during the 72-hour test period, whereas the growth rates of algae exposed to a TWA concentration of 16 and 82 mg/L were increasingly reduced. Statistically significant inhibition of growth rate was found at a TWA concentration of 16 and 82 mg/L.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 Under the conditions of the present study with Pseudokirchneriella subcapitata, test substance reduced growth rate and inhibited the yield of this fresh water algae species significantly at time weight average exposure concentrations of 16 and 82 mg/L. The EC50 for growth rate inhibition (72 h-ERC50) was at a TWA concentration of 55 mg/L with a 95% confidence interval ranging from 47 to 63 mg/L. The EC10 for growth rate inhibition (72h-ERC10) was at a TWA concentration of 13 mg/L with a 95% confidence interval ranging from 8.9 to 17 mg/L. 72 h-NOEC for growth rate was at a TWA concentration of 3.2 mg/L. As the ratio NOEC/LOEC is higher than 3.2, the NOEC for algae test is considered non acceptable.

Description of key information

OECD Guideline 201, GLP, key study, validity 2:

72h-ErC50 (Pseudokirchneriella subcapitata) =55 mg/L (95% CI: 47 - 63 mg/L) based on time-weight average

72h-ErC10 (Pseudokirchneriella subcapitata) = 13 mg/L (95% CI: 8.9 - 17 mg/L) based on time-weight average concentration

Key value for chemical safety assessment

EC50 for freshwater algae:
55 mg/L
EC10 or NOEC for freshwater algae:
13 mg/L

Additional information

One key study is available (WIL, 2015) to assess the influence of the registered substance on the growth of the freshwater green algae species Pseudokirchneriella subcapitata in a 72h static condition, according to OECD Guideline 201 with GLP statement.

The algae were exposed to test substance at concentrations of 0.032, 0.16, 0.8, 4.0, 20 and 100mg/L. The study was conducted under static conditions with an initial cell density of 104 cells/mL and the total exposure period was 72 hours. Three replicates were tested for each test substance concentration and six replicates for the control. The environmental conditions were within the acceptable limits. Samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and after 72 hours of exposure (GC method).

At the start of the test, the actual test concentrations were in agreement with nominal except at the lowest concentration, for which the actual concentration represented 155% of nominal. At the end of the test, actual concentrations at 0.032 and 0.16 mg/L were below the limit of detection. At 0.8 mg/L the concentration decreased to 55% of initial. At 4.0, 20 and 100 mg/L the concentrations remained stable (80-100% of initial), except for the sample taken from vessel 2 of 20 mg/L (52% of initial). For 20 mg/L at the end of the exposure a weighted average concentration was calculated. Since, all concentrations, except at 100 mg/L, deviated from the nominal at the end of the test the Time weight Average concentrations were calculated to correspond with 0.52, 3.2, 16 and 82 mg/L for a concentration of 0.8, 4.0, 20 and 100 mg/L, respectively. At 0.032 and 0.16 mg/L no TWA concentration could be determined; these concentrations were not needed to determine the EC50 values.

Growth rates were in the range of the controls at the TWA concentrations up to a TWA concentration of 3.2 mg/L during the 72-hour test period, whereas the growth rates of algae exposed to a TWA concentration of 16 and 82 mg/L were increasingly reduced. Statistically significant inhibition of growth rate was found at a TWA concentration of 16 and 82 mg/L.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 Under the conditions of the present study with Pseudokirchneriella subcapitata, test substance reduced growth rate and inhibited the yield of this fresh water algae species significantly at time weight average exposure concentrations of 16 and 82 mg/L. The EC50 for growth rate inhibition (72 h-ERC50) was at a TWA concentration of 55 mg/L with a 95% confidence interval ranging from 47 to 63 mg/L. The EC10 for growth rate inhibition (72h-ErC10) was at a TWA concentration of 13 mg/L with a 95% confidence interval ranging from 8.9 to 17 mg/L. 72 h-NOECr for growth rate was at a TWA concentration of 3.2 mg/L. As the ratio NOEC/LOEC is higher than 3.2, the NOEC for algae test is considered unacceptable.