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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:
From 23 September 2015 to 20 July 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(adopted 23 March 2006) ; Annex 5 corrected 28 July 2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD series on testing and assessment number 23, 2000.
Deviations:
no
GLP compliance:
yes
Remarks:
The certificate copy was not included in the study report.
Test material information:
Composition 1
Specific details on test material used for the study:
APPEARANCE
Reddish solid below 25°C / liquid above 25°C

SOURCE OF TEST MATERIAL
- Source and batch/lot No.of test material: Solvay / MR92143651 / 206887/A
- Expiration date of the batch: 01 January 2017
- Purity/Composition: 86.4%
- Purity/composition correction factor: No correction factor required
- Purity test date: 13 April 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature.
- Stability under test conditions: until 01 January 2017 (expiry date)
- Solubility of the test substance in the vehicle (20°C): Insoluble in Water
- Stability of the test substance in the vehicle (20°C): Not indicated
- Stability at higher temperatures: Yes, maximum temperature: 50°C

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was melted at approximately 40°C before weighing.
- No correction was made for the purity/composition of the test item.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples for possible analysis were taken from all test concentrations and the control.
- Sampling method: One of the filters containing the undissolved test item residue was kept for possible analysis. The method of analysis is described in the field: "Details on analytical methods"
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was melted at approximately 40°C before weighing. Preparation of test solutions started with Water Accommodated Fractions (WAFs) individually prepared at loading rates of 1.0 mg/L and higher. A magnetic stirring period of three days was applied to ensure reaching maximum solubility in test medium. The resulting dispersion was subsequently filtered through a 0.45 μm membrane filter (Whatman; RC55) to remove undissolved test item. Test concentrations below 1 mg/L were prepared by subsequent dilutions of the WAF prepared at 1.0 mg/L in test medium. The final test solutions were all clear and colourless. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 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: Green alga, Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Hardness (Ca+Mg): 0.24 mmol/L
Test temperature:
During the exposure period the temperature measured in the incubator was maintained between 21 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).
pH:
Table I (See field "Any other information on materials and methods incl. tables") 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).
Dissolved oxygen:
Unspecified
Salinity:
Not applicable
Conductivity:
Unspecified
Nominal and measured concentrations:
WAFs prepared at 0.1, 0.32, 1.0, 3.2 and 10 mg/L. However, Analysis showed that measured test concentrations gradually decreased during the final test in all test groups. Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated ; It corresponded with 0.0025, 0.0082, 0.058, 0.086 and 0.29 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution.
- Medium: formulated using Milli-RO water and with 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 50 mg/L ; Hardness (Ca+Mg) 0.24 mmol/L]
- Initial cell density: 1 x 10^4 cells/mL.
- No. of vessels per concentration: 3 replicates.
- No. of vessels per control: 6 replicates.
- Others replicates: - 1 or 2 replicates of each test concentration without algae.
- 1 extra replicate of each test group for sampling purposes.

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water.
- Culture medium different from test medium: No
- Intervals of water quality measurement:
* 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.

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: Unspecified
- Illumination: Continuously
- Light intensity and quality: TLD-lamps with a light intensity within the range of 67 to 77 μE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Shimadzu Spectrophotometer UV-1800 including UVProbe 2.33 software (Shimadzu, Kyoto, Japan).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: Yes
- Results used to determine the conditions for the definitive study: The final test was performed based on the results obtained in this preceding static combined limit/range-finding test (Control + WAFs prepared at 1 and 10 mg/L).
- Test concentrations: Algal cultures were exposed to WAFs prepared at 1.0, 3.2 and 10 mg/L and to dilutions representing 10 and 32% of the 1.0 mg/L WAF. Analysis showed that measured test concentrations gradually decreased during the final test in all test groups. Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated. The range tested based on TWA concentrations corresponded with 0.0025, 0.0082, 0.058, 0.086 and 0.29 mg/L.
Reference substance (positive control):
yes
Remarks:
The reference item was Potassium Dichromate (K2Cr2O7)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.1 mg/L
Nominal / measured:
meas. (TWA)
Basis for effect:
growth rate
Remarks on result:
other: with a 95% confidence interval ranging from 0.092 to 0.12 mg/L.
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.045 mg/L
Nominal / measured:
meas. (TWA)
Basis for effect:
growth rate
Remarks on result:
other: with a 95% confidence interval ranging from 0.035 to 0.053 mg/L.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.008 mg/L
Nominal / measured:
meas. (TWA)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.052 mg/L
Nominal / measured:
meas. (TWA)
Basis for effect:
other: Yield
Remarks on result:
other: with a 95% confidence interval ranging from 0.047 to 0.056 mg/L.
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.025 mg/L
Nominal / measured:
meas. (TWA)
Basis for effect:
other: Yield
Remarks on result:
other: with a 95% confidence interval ranging from 0.019 to 0.029 mg/L.
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.008 mg/L
Nominal / measured:
meas. (TWA)
Basis for effect:
other: Yield
Details on results:
- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): No
- Other:
Measured test concentrations decreased gradually during the final test in all test groups. Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated (See table II in field: "Any other information on results incl. tables".). The range tested based on TWA concentrations corresponded with 0.0025, 0.0082, 0.058, 0.086 and 0.29 mg/L.
Growth rates were in the range of the controls at TWA concentrations of 0.0025 and 0.0082 mg/L during the 72-hour test period, whereas the growth rate of algae exposed to TWA concentrations of 0.058 mg/L and higher were increasingly reduced. Statistically significant inhibition of growth rate was found at TWA concentrations of 0.058 mg/L and higher.
Inhibition of yield increased with increasing TWA concentration of 1-Phenyldecane-1,3-dione from 0.058 mg/L upwards resulting in 99% inhibition at 0.29 mg/L. Statistically significant inhibition of yield was found at TWA concentrations of 0.058 mg/L and higher. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a TWA concentration of 0.058 mg/L when compared to the control.
Results with reference substance (positive control):
- Results with reference substance valid? The historical ranges for growth rate and yield inhibition lie between 0.82 and 2.6 mg/L & 0.20 and 1.1 mg/L, respectively. The observed 72h-ErC50 and 72h-EyC50 for the algal culture tested correspond with this range.

- Overview of % inhibition of growth rate and yield in the reference test: See tables III and IV in field: "Any other information on results incl. tables".

- EC50:
* 72h-EyC50 was 0.43 mg/L with a 95% confidence interval ranging from 0.42 to 0.44 mg/L.
* 72h-ErC50 was 1.0 mg/L with a 95% confidence interval ranging from 0.97 to 1.0 mg/L.
Reported statistics and error estimates:
Normal-distribution and variance-homogeneity of data were tested using the Shapiro Wilk`s and Levene´s tests. No departures from normality or variance homogeneity were detected. Normal-distribution and variance-homogeneity requirements are fulfilled.
Inhibition of growth rate or inhibition of yield were then statistically analyzed comparing the data obtained for the test loading rates compared with those obtained in the blank control using the Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm and the Williams Multiple Sequential t-test Procedure, respectively.
Table II: Measured concentrations versus loading rates
1-Phenyldecane-1,3-dione
(WAF; mg/L)
Measured concentration (mg/L) TWA (mg/L)
t=0h t=24h t=72 h
0.10* 0.0157 0.0018 0.00060 0.0025
0.32* 0.0523 0.0075 0.00074 0.0082
1.0 0.179 0.0652 0.0172 0.058
3.2 0.384 0.0627 0.0423 0.086
10 0.739 0.333 0.109 0.29
* Dilutions of the WAF prepared at 1.0 mg/L

Table III: Overview of % inhibition of growth rate in the reference test:
Nominal conc.
K2Cr2O7 (mg/L)
Mean Std. Dev. n %Inhibition
Control 1.637 0.0382 3  
0.18 1.619 0.0347 3 1.1
0.32 1.533 0.0494 3 6.4
0.56 1.221 0.0263 3 25.4
1.0 0.801 0.0943 3 51.1
1.8 0.388 0.0038 3 76.3
3.2 0.185 0.0714 3 88.7
* - effect was statistically significant

Table IV: Overview of % inhibition of yield in the reference test:
Nominal conc.
K2Cr2O7 (mg/L)
Mean Std. Dev. n %Inhibition
Control 135.5 15.16 3  
0.18 128.0 13.04 3 5.5
0.32 99.2 15.28 3 26.8
0.56 38.1 3.03 3 71.9
1.0 10.4 3.43 3 92.3
1.8 2.2 0.04 3 98.4
3.2 0.8 0.40 3 99.4

Table V shows group mean growth rates and the percentages of growth rate inhibition whereas Table VI shows the mean yields and the percentages of yield inhibition:

Table V: Percentage inhibition of growth rate during the final test

1-Phenyldecane-1,3-dione
TWA conc. (mg/L)
Mean Std. Dev. n %Inhibition
Control 1.749 0.0230 6  
0.0025 1.742 0.0186 3 0.4
0.0082 1.748 0.0236 3 0.0
0.058 1.511 0.0741 3 13.6*
0.086 0.987 0.0870 3 43.6*
0.29 0.159 0.2760 3 90.9*
* - effect was statistically significant

Table VI: Percentage inhibition of yield during the final test
1-Phenyldecane-1,3-dione
TWA conc. (mg/L)
Mean Std. Dev. n %Decrease
Control 189.1 13.30 6  
0.0025 185.2 10.49 3 2.0
0.0082 188.6 13.56 3 0.3
0.058 93.5 22.02 3 50.5*
0.086 18.8 5.38 3 90.1*
0.29 1.1 1.84 3 99.4*
* - effect was statistically significant
Validity criteria fulfilled:
yes
Remarks:
OECD validity criteria, see field "overall remarks"
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, 1-Phenyldecane-1,3-dione reduced growth rate and inhibited the yield of this fresh water algae species significantly at TWA concentrations of 0.058 mg/L and higher.
The EC50 for growth rate inhibition (72h-ErC50) was 0.10 mg/L with a 95% confidence interval ranging from 0.092 to 0.12 mg/L.
The EC50 for yield inhibition (72h-EyC50) was 0.052 mg/L with a 95% confidence interval ranging from 0.047 to 0.056 mg/L.
The 72h-NOEC for both growth rate inhibition and yield inhibition equaled a TWA concentration of 0.0082 mg/L.
Executive summary:

Exposure of Pseudokirchneriella subcapitatata to 1 -Phenyldecane-1,3 -dione was performed according to the OECD Guideline 201 (Alga, Growth Inhibition Test) and GLP statement.

Based on a range-finding test, exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at 1.0, 3.2 and 10 mg/L and to dilutions representing 10 and 32% of the 1.0 mg/L WAF. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Analysis showed that measured test concentrations gradually decreased during the final test in all test groups. Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated. The range tested based on TWA concentrations corresponded with 0.0025, 0.0082, 0.058, 0.086 and 0.29 mg/L.

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

The effect parameters obtained in this study, based on TWA concentrations, are summarized below:

-         72h-ErC50 = 0.10 mg/L,

-         72h-ErC10 =0.0454mg/L &

-         72h-NOEC =0.0082 mg/L.

Description of key information

One study performed according to OECD Guideline 201 (Alga, Growth Inhibition Test) is available on Pseudokirchneriella subcapitata. That key study was carried out without major deviation to the guideline and according to GLP.

Based on a range-finding test, the following WAFs were prepared for the definitive exposure: 0.1, 0.32, 1.0, 3.2 and 10 mg test item/L. Analysis showed that measured test concentrations gradually decreased during the final test in all test groups. Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated. The range tested based on TWA concentrations corresponded with 0.0025, 0.0082, 0.058, 0.086 and 0.29 mg/L.

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

The effect parameters obtained in this study, based on TWA concentrations, are summarized below:

-         72h-ErC50 = 0.10 mg/L,

-         72h-ErC10 =0.0454mg/L &

-         72h-NOEC =0.0082 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
0.1 mg/L
EC10, LC10 or NOEC for freshwater algae:
0.045 mg/L

Additional information