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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:
2016-12-18 to 2016-12-25
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch no. 41251
- Expiration date of the lot/batch: 01 March 2017 (nominal expiry date) (taken from label)
- Purity test date: 2016-12-19 to 2016-12-22

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: until 01 March 2017 (nominal expiry date) (taken from label)

FORM AS APPLIED IN THE TEST (if different from that of starting material): Colourless liquid

-OTHER:

Molecular weight: 320
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Vapour Pressure: 121 mmHg at 25°C
Specific gravity/density: 1.67 at 25°C

Solubility in water: Practically insoluble
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.10, 1.0, 10 and 100% of a SS at 100 mg/L
- Sampling method: 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: 2.0 mL
Storage: Not applicable, samples were added to a 10 mL vial containing 40 µL dimethyl sulfoxide (DMSO) and analyzed on the day of sampling.

At the start of the test additional smaller samples were taken from the two highest test concentrations, i.e. 1.0 mL from the 10% SS and 100 µL from the 100% SS, and added to 10 mL vials containing 40 µL DMSO. At the end of the exposure period, samples were taken from one replicate of each concentration.

- Sample storage conditions before analysis: Not applicable, samples were added to a 10 mL vial containing 40 µL dimethyl sulfoxide (DMSO) and analyzed on the day of sampling.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The batch of AsahiklinTM AC-2000 tested was a colourless liquid with a purity of 100.00% and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.

Preparation of test solutions started with a loading rate of 100 mg/L applying one day of slow magnetic stirring in a closed vessel to reach the maximum dissolution of the test item in the test medium. The resulting aqueous mixture was left to stabilize for one hour where after the clear and colourless saturated solution (SS) was siphoned out and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. 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 density of 10^4 cells/mL.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): Not specified
- Method of cultivation: 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.

ACCLIMATION
- Acclimation period: not specified
- Culturing media and conditions (same as test or not): Same
Test type:
static
Water media type:
other: Test Medium
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
Between 22 and 23°C
pH:
Control
t = 0 h = 7.4
t = 72 h = 7.7

Test material (100% SS at 100 mg/L)
t = 0 h = 7.3
t = 72 h = 7.8
Nominal and measured concentrations:
0.10, 1.0, 10 and 100% of a SS at 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 120 mL, airtight closed with no headspace to prevent any loss of the test item due to volatilization.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 120 mL; no headspace
- Aeration: Not specified
- Renewal rate of test solution (frequency/flow rate): Not specified
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density:
- No. of organisms per vessel: 3 days before the start of the 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.
- No. of vessels per concentration (replicates): 6 each for the highest test concentration; 3 each lower test concentration; 1 extra replicate of each test concentration for sampling purposes; 1 or 2 replicates of each test concentration without algae.
- No. of vessels per control (replicates): 6 each for the control; 1 extra replicate of the control for sampling purposes

GROWTH MEDIUM
- Standard medium used: Yes
- Detailed composition if non-standard medium was used: Please see details below:

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) with the following composition:

NaNO3: 500 mg/L
K2HPO4.3H2O: 52 mg/L
MgSO4.7H2O: 75 mg/L
Na2CO3.10H2O: 54 mg/L
C6H8O7.H2O: 6 mg/L
NH4NO3: 330 mg/L
CaCl2.2H2O: 35 mg/L
C6H5FeO7.xH2O: 6 mg/L
H3BO3: 2.9 mg/L
MnCl2.4H2O: 1.81 mg/L
ZnCl2: 0.11 mg/L
CuSO4.5H2O: 0.08 mg/L
(NH4)6Mo7O24.4H2O: 0.018 mg/L

Pre-culture medium: Adjusted M2; Standard M2 according to the OECD 201 Guideline but with a larger amount of NaHCO3, the addition of HEPES buffer and a lower pH. This medium will be formulated using Milli-RO water and will have the following composition:

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: 64 µg/L
Na2EDTA.2H2O: 100 µg/L
H3BO3: 185 µg/L
MnCl2.4H2O: 415 µg/L
ZnCl2: 3 µg/L
CoCl2.6H2O: 1.5 µg/L
CuCl2.2H2O: 0.01 µg/L
Na2MoO4.2H2O: 7 µg/L
NaHCO3: 300 mg/L
HEPES buffer: 6 mmol/L
Hardness (Ca+Mg): 0.24 mmol/L (24 mg CaCO3/L)
pH: 7.1 ± 0.3

OTHER TEST CONDITIONS
- Sterile test conditions: not specified
- Adjustment of pH: yes
- Photoperiod: Continuous
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 87 to 90 µE.m^-2.s^-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; spectrophotometer]: 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 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval. Cell densities were recorded at 24-hour intervals in the control and the highest test concentration. Lower concentrations were measured only at the end of the exposure period.
- Other: Appearance of cells: At the end of the test microscopic observations were performed on the highest test concentration and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Range finding study: Yes
- Test concentrations: A combined limit/range-finding test was performed, exposing six replicates of exponentially growing algal cultures to a control and to 100% of the SS. In addition, three replicates per group were exposed to 0.10, 1.0 and 10% of the SS.
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.098 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.098 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.098 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.098 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
Measured test item concentrations:

Analysis of the samples taken from 10 and 100% of the SS at the start of the test showed measured concentrations of 0.047 and 0.72 mg/L, respectively. The concentration of the 10% SS solution decreased below the concentration of the lowest calibration solution, i.e. below 0.008 mg/L, within 24 hours of exposure. The concentration of the 100% SS solution decreased to 13% of the initial concentration after 24 hours of exposure and decreased further below the concentration of the lowest calibration solution at the end of the test. The initial measured concentrations in the samples taken from 0.10 and 1.0% of the SS were below the concentration of the lowest calibration solution. Given these analytical results and the biological results as described in section 7.2 of the attached study report, the effect parameters were based on the TWA concentration of the 100% SS that was calculated to be 0.098 mg/L.

Measured concentrations in the samples taken from the 100% SS solution with and without algae were similar, indicating that the presence of algal cells did not affect the test item concentration.

Mean cell densities
Figure 1 in the attached study report shows growth curves at different concentrations of AsahiklinTM AC-2000. The individual and group mean cell densities measured at 24h intervals are given in Table 6 of the attached study report.

Inhibition of growth rate and inhibition of yield:

Table 1 shows group mean growth rates and the percentages of growth rate inhibition (total test period) whereas Table 2 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized in Table 3

Statistical analysis of the data was not performed because cell growth in the highest test concentration was similar to the control. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the highest test concentration when compared to the control.

Determination of effect concentrations:

Table 4 shows the effect parameters based on TWA concentrations.

Experimental conditions:
Table 5 shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (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 22and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: The actual responses in the reference test with K2Cr2O7 were within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was within the expected range of 0.28 to 0.9 mg/L. Hence, the sensitivity of the daphnia was within the range determined with the historical data collected at Charles River Den Bosch.The 24h-EC50 was 0.91 mg/L with a 95% confidence interval ranging from 0.77 to 1.1 mg/L. The 48h-EC50 was 0.58 mg/L with a 95% confidence interval ranging from 0.49 to 0.67 mg/L.

Table 1. Percentage inhibition of growth rate (total test period)

AsahiklinTMAC-2000, % SS at 100 mg/L

Mean

S.D.

n

%Inhibition

Control

1.604

0.0544

6

 

0.10

1.570

0.0428

3

2.1

1.0

1.540

0.0673

3

4.0

10

1.578

0.0487

3

1.6

100 (0.098)

1.614

0.0306

6

-0.6

( ) TWA concentration (mg/L)

 

Table 2. Percentage inhibition of growth rate at different time intervals

 

AsahiklinTM AC-2000, % SS at

100 mg/L

 

 

n

 

0 – 24 h

 

24 – 48 h

 

48 – 72 h

Mean

 

%Inhibition

Mean

 

%Inhibition

Mean

 

%Inhibition

Control

6

1.427

0.0

1.877

0.0

1.509

0.0

100 (0.098)

6

1.684

-18.0

1.521

19.0

1.637

-8.5

( ) TWA concentration (mg/L)

 

Table 3. Percentage inhibition of yield

AsahiklinTM AC-2000, % SS at 100 mg/L

Mean

S.D.

n

%Inhibition

Control

123.4

19.04

6

 

0.10

110.8

14.76

3

10.2

1.0

101.8

19.75

3

17.5

10

113.5

15.98

3

8.0

100 (0.098)

126.2

11.76

6

-2.2

( ) TWA concentration (mg/L)

 

Table 4. Effect parameters

Parameter (mg/L)

NOEC

EC50

Growth Rate

Value

0.098

>0.098

Yield

Value

0.098

>0.098

 

Table 5. pH levels

AsahiklinTM AC-2000, % SS at 100 mg/L

pH

t = 0 h

t = 72 h

Control

7.4

7.7

100 (0.098)

7.3

7.8

( ) TWA concentration (mg/L)

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, no inhibition of both growth rate and yield was recorded in a saturated solution prepared at a loading rate of 100 mg AsahiklinTM AC-2000 per litre. Due to the low solubility of AsahiklinTM AC-2000in water, concentration levels that might be toxic for algae could not be reached.

The 72h-EC50 for growth rate inhibition (ERC50) and for yield inhibition (EYC50) was above a TWA concentration of 0.098 mg/L.

The 72h-NOEC for inhibition of both growth rate and yield was 0.098 mg/L.
Executive summary:

In a key guideline (OECD 201) study, the fresh water algal growth inhibition potential of the test material (AsahiklinTM AC-2000) was evaluated using Pseudokirchneriella subcapitata.

 

The test material was a colourless liquid with a purity of 100.00% and not completely soluble in test medium at the loading rate initially prepared. Preparation of test solutions started with a loading rate of 100 mg/L applying one day of slow magnetic stirring in a closed vessel to reach the maximum dissolution of the test item in the test medium. The resulting aqueous mixture was left to stabilize for one hour where after the clear and colourless saturated solution (SS) was siphoned out and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium.

 

A combined limit/range-finding test was performed, exposing six replicates of exponentially growing algal cultures to a control and to 100% of the SS. In addition, three replicates per group were exposed to 0.10, 1.0 and 10% of the SS. The initial algal cell density was 104 cells/mL and the total exposure period was 72 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure. Analysis of the samples taken from 10 and 100% of the SS at the start of the test showed measured concentrations of 0.047 and 0.72 mg/L, respectively. The concentration of the 10% SS solution decreased below the concentration of the lowest calibration solution, i.e. below 0.008 mg/L, within 24 hours of exposure. The concentration of the 100% SS solution decreased to 13% of the initial concentration after 24 hours of exposure and decreased further below the concentration of the lowest calibration solution at the end of the test. The initial measured concentrations in the samples taken from 0.10 and 1.0% of the SS were below the concentration of the lowest calibration solution. Given these analytical results and the biological results the effect parameters were based on the TWA concentration of the 100% SS that was calculated to be 0.098 mg/L.

 

Under the conditions of the study with Pseudokirchneriella subcapitata, no inhibition of both growth rate and yield was recorded in a saturated solution prepared at a loading rate of 100 mg AsahiklinTM AC-2000 per litre. Due to the low solubility of the test material in water, concentration levels that might be toxic for algae could not be reached.

 

The 72h-EC50 for growth rate inhibition (ERC50) and for yield inhibition (EYC50) was above a TWA concentration of 0.098 mg/L.

 

The 72h-NOEC for inhibition of both growth rate and yield was 0.098 mg/L.

Description of key information

The 72h-EC50 for growth rate inhibition (ERC50) and for yield inhibition (EYC50) was determined to be above a TWA concentration of 0.098 mg/L.

 

The 72h-NOEC for inhibition of both growth rate and yield was determined to be 0.098 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
0.098 mg/L

Additional information

In a key Guideline (OECD 201) fresh water algal growth inhibition test (Charles River Laboratories Den Bosch BV., 2017c), Pseudokirchneriella subcapitata was exposed to the test material (1,1,1,2,2,3,3,4,4,5,5,6,6-tridecafluorohexane dissolved in a test medium) at concentrations of 0.10, 1.0, 10 and 100% of a saturated solution at 100 mg/L for a period of 72 hours.

 

Under the conditions of the study, no inhibition of both growth rate and yield was recorded. Due to the low solubility of the test material in water, concentration levels that might be toxic for algae could not be reached.

 

The 72h-EC50 for growth rate inhibition (ERC50) and for yield inhibition (EYC50) was above a TWA concentration of 0.098 mg/L.

 

The 72h-NOEC for inhibition of both growth rate and yield was 0.098 mg/L.