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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:
2017
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
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Product description: Potassium isooctadecanoate/potassium isostearate
Name: Isooctadecanoic acid, potassium salt
CAS No.: 66469-15-6
Physical state: off white to yellowish solid at 20 °C
Batch No.: PFS-755-169
Re-certification date of batch: 12 December 2017
Purity: 100 % (UVCB)
pH, 10% in DI water 8.0 - 9.0
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light
Analytical monitoring:
yes
Details on sampling:
Samples were taken in 3 replicates from three test concentrations (6.25 mg/l, 25.0 mg/l and 100 mg/l) and the control (NC) at 0 h, 24 h, 48 h and 72 h from the separate test vessels without algae. For stabilisation of the sample 2 mL acetonitrile were added into the 4 mL glass vials. With every sampling 2 mL test-solution were taken and filled into 4 mL glass vials. The pipette tip was rinsed with the test solution-acetonitril-mixture in the glass vial two or three times. Two of the replicates were stored at 5°C +/- 3°C until being sent to the analytical laboratory. The third retain samples were kept in the freezer until finalization of the study. Proper departure and arrival of the samples were documented.
Vehicle:
no
Details on test solutions:
According to the observations and results obtained in the range-finding test, the following five nominal test concentrations with a spacing factor of 2 were tested: 6.25 mg/L, 12.5 mg/L, 25.0 mg/L, 50.0 mg/L and 100.0 mg/L.
The test item has a low solubility of < 1 mg/L. Therefore a saturated stock solution with a loading rate of 100 mg/L was prepared and stirred 24 hours for a maximum solubility. To obtain a nominal loading rate of 100 mg/L after addition of the algal inoculum, the saturated stock solution was prepared factor 1.11 higher to take the dilution of the test solution by the algal inoculum into account. 111.4 mg of the test item was weighed and transferred into 1000 mL of algal medium. The stock solution and also algal medium for the controls were stirred for 24 h with an overhead shaker (about 15 rpm). The temperature was between 25.6 to 26.0°C. After stirring and a sedimentation period of 30 min., 550 mL of the saturated stock solution was withdrawn from the middle of the flask and transferred into a 1000 mL-beaker. This stock solution was used for the preparation of the test vessels with different loading rates.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Tests were carried out with the green alga Raphidocelis subcapitata (Strain No. 61.81 SAG) from the Culture Collection of Algae at the University of Goettingen and cultivated in suspension culture. The strain used for this study has been cultured in the laboratory of Hydrotox GmbH since November 2016. Twice a week, the stock suspension is diluted into fresh Holm-Hansen medium under axenic conditions to keep it in exponential growth.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Algal medium according to OECD 201.
Test temperature:
The temperature in the test was in the range of 22.2 to 23.1°C, which is within the requirements of the guideline (21-24°C). The variation in temperature was 0.9 K, which was within the required range of +/- 2 K.
pH:
The pH-values were measured at the beginning and at the end of the test (after 72 h). The pH changed not more than 0.4 units during the test.
Dissolved oxygen:
not specified
Salinity:
According to algal medium according to OECD 201.
Conductivity:
Algal medium according to OECD 201.
Nominal and measured concentrations:
In the control, the test item was not detected. The initial measured test item concentrations in the test item treatments were < LOQ – 7.71 mg/L at the start of the test (0h). Until the end of the exposure period, the measured test item concentrations decreased to < LOQ (72 h).
Due to the fact that only three of the five test item concentrations were measured within the chemical analysis and the results of the
measurement showed a high variability, it is not feasible to conduct the data evaluation based on the measured test item concentrations. All results are given in relation to the nominal test item concentrations.
Details on test conditions:
Conditions of exposure

The test and control vessels were sealed with a sterile cellulose stopper and incubated in a light incubator. The incubator provides stable temperature and light conditions. The test vessels were placed on a rotary disc made of acrylic glass, which provides space for 21 vessels on each of the four floors. The rotation of about 2 revolutions per minute (rpm) ensures that each vessel is exposed to equal light conditions. Each rotation includes 5 sudden stops by which the test suspensions are shaken. The flasks are illuminated laterally by eight fluorescent tubes (58 Watt each), which are separated from the incubation chamber by heat absorbing glass. Mean light intensity was controlled by light intensity measurements with a spherical sensor on each of the four floors. The min/max temperature in a reference flask was recorded on a daily basis. The increase in algal biomass was monitored by measuring the chlorophyll fluorescence in each test vessel after 0, 24, 48 and 72 hours. At the end of the test, the pH was measured in one flask from each nominal concentration.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
63.43 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: (95%-CI: 35.03 -112.71 mg/L)
Key result
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
33.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: (95%-CI: 20.92 - 53.80 mg/L)
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
23.93 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Remarks on result:
other: (95%-CI: 14.59 - 39.24 mg/L)
Key result
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
32.79 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
cell number
Remarks on result:
other: (95%-CI: 12.07 - 87.38 mg/L)
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
22.23 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
cell number
Remarks on result:
other: (95%-CI: 9.85 - 50.22 mg/L)
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
18.14 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
cell number
Remarks on result:
other: (95%-CI: 7.65 - 42.99 mg/L)
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
cell number
Results with reference substance (positive control):
Potassium dichromate served as positive control in order to assess the validity of the test system. The last quality check was performed in February 2017.
The ErC50 was 0.95 mg/L (95%-Cl: 0.61 – 1.47 mg/L) and the EyC50 (Yield) was 0.06 mg/L (95%-CI: 0.00 – 1.29 mg/L).
Reported statistics and error estimates:
The data was evaluated using the statistical software ToxRat Professional (Version 3.2.1, ToxRat Solutions GmbH, Alsdorf).

Results

The Algae Growth Inhibition Test was performed with five loading rates between 6.25 mg/L and 100 mg/L. Because of the low solubility of the test item, resulting in a very low recovery rate, all results are given in relation to the nominal test item concentrations. The following results were obtained:

ELRx values determined with the nominal concentrations:

Growth Rate:

ErLR50 : 63.43 mg/L (95%-CI: 35.03 -112.71 mg/L)

ErLR20 : 33.44 mg/L (95%-CI: 20.92 - 53.80 mg/L )

ErLR10 : 23.93 mg/L  (95%-CI: 14.59 - 39.24 mg/L)

NOErLR : = 25.00 mg/L

LOErLR : = 50.00 mg/L

Yield:

EyLR50 : 32.79 mg/L (95%-CI: 12.07 - 87.38 mg/L)

EyLR20 : 22.23 mg/L (95%-CI: 9.85 - 50.22 mg/L)

EyLR10 : 18.14 mg/L  (95%-CI: 7.65 - 42.99 mg/L)

NOEyLR : = 25.00 mg/L

LOEyLR  : = 50.00 mg/L

Mean growth rates and mean yields and their inhibitions compared to the control after 72 h of exposure

   Control  A  B  C  D  E
 Nominal test concentrations [mg/l]  0  6.25  12.5  25.0  50.0  100.0
 Number of replicates  6  3  3  3  3  3
 Mean growth rate  1.49  1.53  1.60  1.38  0.90  0.46
 Mean yield  6.38  8.18  8.55  4.92  1.53  0.21
 Mean inhibition of growth rate [%]  -  -2.9*  -7.6  -7.3 39.5  69.4
  Mean inhibition of yield [%]  -  -28.1*  -33.8  22.9  76.0  96.8

* negative inhibition means growth promotion

The morphology of the algal cells in the five test solutions as well as in the control showed no abnormality.

Validity criteria

The biomass in the controls increased within 72 h from 0.07 x 10^5 algal cells/mL to 6.457 x 10^5 (factor 89), which is within the validity range of the test (required ≥ 16). The maximum mean variation coefficient of the growth rate from day to day (day 0-1, 1-2 and 2-3) was 20.7 % in the controls, which is within the validity range of 0 – 35 %. The variation coefficient calculated for the mean growth rate from day 0 to day 3 was 4.6 % and below the upper limit of 7%.

Additional criteria mentioned in the guidelines

Light intensity during the test was 70.7 µE/m2 s +/- 5.3 % (PAR), which was within the required range of 60 – 120 µE m-2 s +/- 15 %. The temperature in the test was in the range of 22.2 to 23.1°C, which is within the requirements of the guideline (21-24°C). The variation in temperature was 0.9 K, which was within the required range of +/- 2 K.

Validity criteria fulfilled:
yes
Conclusions:
An algae, growth inhibition test with Raphidocelis subcapitata was performed with five loading rates between 6.25 mg/L and 100.0 mg/L of the test item. Because of the low solubility of the test item, resulting in a very low recovery rate, all results are given in relation to the nominal test item concentrations. The following results were obtained:

Growth rate:
ErLR50 : 63.43 mg/L (95%-CI: 35.03 -112.71 mg/L)

Yield:
EyLR50 : 32.79 mg/L (95%-CI: 12.07 - 87.38 mg/L)
Executive summary:

In a Klimisch 1 algae, growth inhibition GLP study with Raphidocelis subcapitata five loading rates between 6.25 mg/L and 100.0 mg/L were analyzed. Because of the low solubility of the test item, resulting in a very low recovery rate, all results are given in relation to the nominal test item concentrations. The following results were obtained:

Growth rate:

ErLR50 : 63.43 mg/L (95%-CI: 35.03 -112.71 mg/L)

Yield:

EyLR50 : 32.79 mg/L (95%-CI: 12.07 - 87.38 mg/L)

Description of key information

An algae, growth inhibition test with Raphidocelis subcapitata was performed with five loading rates between 6.25 mg/L and 100.0 mg/L of the test item. Because of the low solubility of the test item, resulting in a very low recovery rate, all results are given in relation to the nominal test item concentrations. The following results were obtained:

Growth rate:

ErLR50 : 63.43 mg/L (95%-CI: 35.03 -112.71 mg/L)

Key value for chemical safety assessment

EC50 for freshwater algae:
63.43 mg/L

Additional information

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