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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:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
Name: 9-Octadecenoic acid (9Z)-, propyl ester
Product Description: Propyl oleate
CAS No.: 111-59-1
Physical state: pale yellow to yellow liquid at 20 °C
Batch No.: 37584
Re-certification date of batch: 08 March 2018
Purity: > 99 % (fatty acid propyl esters, max water content 0.3 % (w/w))
Free Fatty Acid, % Oleic: 0.1
Color, Gardner: 3
Iodine Value, cg I2/g 75
Cloud Point, degrees C 0
Saponification Number, mg KOH/g 177
Moisture, % 0,07
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light
Analytical monitoring:
yes
Remarks:
HPLC method
Details on sampling:
Chemical analysis of the test item to determine exposure concentration and stability of the test item during test duration was performed at the test site biochemA, Im Oberwald 19b, D-79359, Riegel a.K., Germany.
Samples were taken in duplicate from the separate test vessels and control without algae at 0 h, 24h, 48 h and 72h. Additionally after 72h samples will be taken in duplicate from the test vessels and control with algae. For stabilisation 4 ml acetonitril was submitted in 8 mL glass vials. With every sampling 1 mL of the test solution was taken and filled into the 8 mL glass vials. This resulted in a dilution of test solution by factor 5. The samples were kept in the freezer at < -18°C. After the experimental phase one sample of every sampling date was sent to the analytical laboratory. The second sample was kept in the freezer as retain sample 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 very 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 1.111 higher to take the dilution of the test solution by the algal inoculum into account. 111.2 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 22 rpm). The temperature was between 21.2 to 23.0°C. After stirring and a sedimentation period of 35 min., 800 mL of the saturated stock solution was withdrawn from the middle of the flask and transferred into a 1000 mL-beaker. The turbid solution was filtered with a nylon filter (0.45 µm, Nalgene Rapid Flow; Lot: 1168567) to prepare a clear test solution. The clear stock solution was used for the preparation of the test vessels with the different loading rates.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test organism Raphidocelis subcapitata (Strain No. 61.81 SAG) originates from the Culture Collection of Algae at the University of Goettingen and was cultivated in suspension culture. The strain used for this study has been cultured at Hydrotox GmbH since July 2017. Twice a week, the stock suspension is inoculated into fresh Holm-Hansen medium (see Appendix I) 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:
The test was performed with Holm-Hansen medium for algal suspension culture.
Test temperature:
The temperature in the test was in the range of 22.7 to 23.0°C, which is within the requirements of the guideline (21-24°C). The variation in temperature was 0.3 K, which was within the required range of +/- 2 K.
pH:
The pH-values were measured at the beginning (7.7 - 7.9) and at the end of the test after 72 h (7.6 - 7.7). The pH changed not more than 0.3 units during the test.
Dissolved oxygen:
not specified
Salinity:
The test was performed with Holm-Hansen medium for algal suspension culture.
Conductivity:
not specified
Nominal and measured concentrations:
The insolubility of the test item, resulting in no detection of the test item. Therefore all results are given in relation to the nominal test item concentrations. The following results were obtained:
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
<= 6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
<= 6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
LOELR
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Results with reference substance (positive control):
Quality assurance takes place at regular intervals by testing the sensitivity of the test organisms to the reference item potassium dichromate. The recent quality testing was performed in August 2017 which resulted in an ErC50 of 1.11 mg/L (CL 95 %: 0.65 - 1.86 mg/L); interlaboratory test result for ErC50: mean of 1.19 mg/L with SD of 0.27 mg/L) and an EyC50 of 0.07 mg/L (CL 95 %: 0.00 – 2.47 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

Summary

The Algae Growth Inhibition Test was performed with five loading rates between 6.25 mg/L and 100 mg/L. The insolubility of the test item, resulting in no detection of the test item. Therefore 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: > 100 mg/

ErLR20: > 100 mg/

ErLR10: > 100 mg/

NOErLR: ≤ 6.25 mg/L

LOErLR: = 6.25 mg/L       

Yield:

EyLR50: > 100 mg/

EyLR20: n.d.

EyLR10: n.d.

NOEyLR: ≤ 6.25 mg/L

LOEyLR: = 6.25 mg/L

The test is valid according to OECD 201 (23 March 2006). Substance-specific chemical analysis was based on the detection of the test item, which has a purity of 100%.

In the control, the test item was not detected. In all test item treatments the test item was below the Limit of Detection (LOD = 0.0310 mg/L).

Growth rate and yield

Inhibitions of the growth rates in relation to the controls were between 3.2% and 7.8%. But no dose-response relationship of the growth rates could be observed. The morphology of the algal cells in the five test solutions as well as in the control showed no abnormality.

 

Effect concentrations

The effect concentrations after 72 h exposure were calculated with the statistical software ToxRat Professional 3.2.1. Because the measurements of the chemical analysis were below the limit of detection, it is not feasible to conduct the data evaluation based on the measured test item concentrations. Therefore all results are given in relation to the nominal test item concentrations. Due the inhibitions of the growth rate was below the EC10 value without a dose-response relationship, a statistical evaluation for ErLRx was not possible. Therefore the EC values are presented as > 100 mg/L. The maximum inhibition of the yield was 33.4%. Therefore the EyLR50 are presented as > 100 mg/L.

Results of accompanying chemical analysis

In the control, the test item was not detected. In all test item treatments the test item could not be detected and was below the limit of detection (LOD = 0.0310 mg/L). Therefore no calculations of the measured concentrations were possible. In a separate Non-GLP trial using on-line column extraction, up to 1.80 mL sample were injected. Due to negative system peak, no reasonable result could be obtained. Additionally samples were evaporated to dryness and reconstituted in a small volume of mobile phase. Analysis of these samples (e.g. conc. 50 mg/l (D) and 100 mg/l (E)) also resulted in “no peak”.

Validity criteria

- The biomass in the controls increased within 72 h from 0.07 x 105 algal cells/mL to 12.724 x 105 (factor 181.8), 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 10.9 % 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 0.6 % and below the upper limit of 7%.

Additional criteria mentioned in the guidelines

- Light intensity during the test was 71.1 µE/m2 s +/- 8.5 % (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 22.7 to 23.0°C, which is within the requirements of the guideline (21-24°C). The variation in temperature was 0.3 K, which was within the required range of +/- 2 K. The test is valid according to OECD 201 (23 March 2006).

Table: 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 concentration (mg/L)  0  6.25  12.5  25.0  50.0  100.0
 Number of replicates  6  3  3  3  3
 Mean growth rate  1.73  1.61  1.61  1.65  1.68  1.60
 Mean yield  12.65  8.63  8.69  9.92  10.68  8.43
 Mean inhibition of growth rate (%)  -  7.3  7.2  5.1  3.2  7.8
 Mean inhibition of yield (%)  -  31.8  31.4  21.6  15.6  33.4
Validity criteria fulfilled:
yes
Conclusions:
The OECD 201 Algae Growth Inhibition Test was performed with five loading rates between 6.25 mg/L and 100 mg/L. The insolubility of the test item, resulting in no detection of the test item. Therefore all results are given in relation to the nominal test item concentrations. The following results were obtained: ErLR50 : > 100 mg/L, ErLR20 : > 100 mg/L, NOErLR : ≤ 6.25 mg/L.
Executive summary:

An Algae Growth Inhibition Test with Raphidocelis subcapitata was conducted according to OECD 201 (23 March 2006) in order to investigate the effect of the test substance on the growth of algae. The OECD 201 Algae Growth Inhibition Test was performed with five loading rates between 6.25 mg/L and 100 mg/L. The insolubility of the test item, resulting in no detection of the test item. Therefore all results are given in relation to the nominal test item concentrations. The following results were obtained: ErLR50 : > 100 mg/L, ErLR20 : > 100 mg/L, NOErLR : ≤ 6.25 mg/L.

Description of key information

The OECD 201 Algae Growth Inhibition Test was performed with five loading rates between 6.25 mg/L and 100 mg/L. The insolubility of the test item, resulting in no detection of the test item. Therefore all results are given in relation to the nominal test item concentrations. The following results were obtained: ErLR50 : > 100 mg/L, ErLR20 : > 100 mg/L, NOErLR : ≤ 6.25 mg/L.

Key value for chemical safety assessment

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

Additional information