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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:
24 Feb 2017 - 07 Apr 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted March 23, 2006; Annex 5 corrected 28 July 2011.
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures
Version / remarks:
OECD series on testing and assessment number 23, 2000
GLP compliance:
yes
Specific details on test material used for the study:
Test item information
Identification: Soybean oil, epoxidized, Me ester, reaction products with propylene glycol
Appearance: Yellow liquid (determined by Charles River DenBosch)
Batch: EM97160711
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 20 July 2017 (expiry date)
Test item: 207811/A
Purity/composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Analytical monitoring:
yes
Remarks:
TOC analysis
Details on sampling:
Single samples for possible analysis of the Total Organic Carbon (TOC) concentration were taken from all test concentrations and the control
according to the schedule below.

Frequency at t=0 h and t=72 h
Volume 40 mL or 50 mL (at t=0h final test only)

Storage Samples were stored in a refrigerator (2-8°C) until analysis. At the end of the exposure period, the extra replicates incubated without algae were pooled at each concentration before sampling. Additionally, reserve samples of 40 mL were taken for possible analysis. If not used, these samples were stored in a refrigerator (2-8°C) 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:
The batch of Soybean oil, epoxidized, Me ester, reaction products with propylene glycol tested was a yellow liquid UVCB substance and not completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item. Preparation of test solutions started with individually prepared loading rates. A two-day magnetic stirring period was applied to ensure reaching maximum solubility of the test item in medium. The obtained mixtures were then allowed to settle for an overnight period. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. Test solutions were clear and colorless at the end of the preparation procedure, except for the WAFs prepared at 32 mg/L and higher that were slightly hazy.

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 104 cells/mL. Any residual volumes were discarded unless otherwise requested by the Study Director.
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
- 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. 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.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
During the exposure period the temperature measured in the incubator was maintained between 21.8 and 22.7°C
pH:
pH t=0h : 7.0-8.1
pH t=72h: 7.4-7.6
Nominal and measured concentrations:
Nominal concentrations: WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L
TOC measurements:
TOC t=0h : 0*, 2.8, 6.4, 11, 16 and 24 mg TOC/L
TOC t=72h: 0.30*, 3.0, 6.0, 9.7, 15 and 19 mg TOC/L
* Values below 1 mg C/L should be considered indicative because of the limit of quantification of the analytical method.
Details on test conditions:
TEST CONCENTRATIONS
Test item WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L.
Controls Test medium without test item or other additives.
Replicates - 6 replicates of the control,
- 3 replicates of each test concentration
- 2 or 3 replicates of each test group without algae

TEST PROCEDURE AND CONDITIONS
Test duration: 72 hours
Test type: Static
Test vessels: 100 mL, all-glass, containing 50 mL of test solution
Medium: M2
Cell density: An initial cell density of 1 x 10^4 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 82 to 87 μE.m^-2.s^-1.
Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

MEASUREMENTS AND RECORDINGS
-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 control and the highest concentration tested to observe for any abnormal appearance of the algae.

EFFECT PARAMETERS MEASURED
Most of the test solutions were so turbid that they disturbed spectrophotometric measurement. Therefore algal density was determined by use of a microscope and a counting chamber throughout the test.

TEST CONCENTRATIONS
Spacing factor for test concentrations: 1.8

Combined Limit/Range-Finding Test:
The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae were exposed to an untreated control and a WAF prepared at a loading rate 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 WAFs prepared at 1.0 and 10 mg/L in the combined range-finding test.
•pH was only measured in the control and the highest test concentration.
•At the end of the test algae were not observed to verify a normal and healthy appearance.
•Cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm).

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:
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:
EL10
Effect conc.:
38.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL (30.4- 45.2)
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
52.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% Cl ( 46.6-58.9)
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
13.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL ( 10.2-17.1)
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
< 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Measured Carbon concentrations remained fairly stable during the 72-hour test period with average Carbon concentrations at levels corresponding to 30-48% relative to the initial loading rates. Since TOC-analysis is a non-specific method, no further estimations for exposure concentrations were performed. TOC-analysis of the samples taken from the WAF prepared at 100 mg/L showed an initial TOC concentration of 26.0 mg C/L. This concentration remained stable during the 48-hour test period with a measured concentration of 24 mg C/L at the end of the test. Based on this result, it could be anticipated that approximately 36 mg/L of the initial loading of 100 mg/L was actually in solution. Further results will consequently be reported in terms of loading rates and expressed in mg per litre.

Growth rates were only marginally reduced in comparison to the controls at the loading rates from 10 to 32 mg/L during the 72-hour test period, whereas the growth rate of algae exposed to loading rates of 56 and 100 mg/L were significantly and increasingly reduced. Statistically significant inhibition of growth rate was found at loading rates of 32 mg/L and
higher. Inhibition of yield increased with increasing loading rate of Soybean oil, epoxidized, Me ester, reaction products with propylene glycol from 14% at the lowest loading rate of 10 mg/L to 75% at the highest loading rate of 100 mg/L. Statistically significant inhibition of yield was found at all loading rates tested. Instead, the EL10 was calculated as an equivalent alternative. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the different loading rates when compared to the control.

Acceptability of the test:
1. In the controls, cell density increased by an average factor of at least 16 within the exposure period (i.e. 117).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 18.2%)
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 2.4%).
Results with reference substance (positive control):
Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/L and higher. The EC50 for growth rate inhibition (72h-ERC50) was 1.2 mg/L with a 95% confidence interval ranging from 1.1 to 1.2 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 0.43 mg/L with a 95% confidence interval ranging from 0.42 to 0.44 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 corresponds with this range.
Reported statistics and error estimates:
For determination of the NOELR and the EL50 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 Procedure, α=0.05, one-sided, smaller). Additionally, the EL10 and EL20 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 ELx values was based on probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield
inhibition versus the logarithms of the corresponding test item loading rates. No EL50-values could be calculated for growth rate (EL50 > maximum loading rate tested). The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Inhibition results

Percentage inhibition of growth rate (total test period) during the final test

 

Test item** Loading rate(mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.585

0.0385

6

10

1.533

0.0452

3

3.3

18

1.526

0.0194

3

3.7

32

1.500

0.0470

3

5.4*

56

1.311

0.0160

3

17.2*

100

1.126

0.1008

3

29.0*

* Effect was statistically significant

**Test item = Soybean oil, epoxidized, Me ester, reaction products with propylene glycol

Percentage inhibition of yield during the final test

Test item** Loading rate

(mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

115.7

14.20

6

10

99.0

13.87

3

14.4*

18

96.4

5.65

3

16.7*

32

89.5

13.22

3

22.7*

56

50.2

2.43

3

56.5*

100

29.2

8.75

3

74.8*

* Effect was statistically significant

**Test item = Soybean oil, epoxidized, Me ester, reaction products with propylene glycol

 

Validity criteria fulfilled:
yes
Remarks:
See details on results
Conclusions:
In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, Soybean oil, epoxidized, Me ester, reaction products with propylene glycol reduced growth rate of this fresh water algae species significantly at loading rates of 32 mg/L and higher.
Executive summary:

A full OECDTG 201 GLP test was performed with Pseudokirchneriella subcapitata. Six exponentially growing algal cultures were exposed for 72h to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L of Soybean oil, epoxidized, Me ester, reaction products with propylene glycol.The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of exposure. Measured Carbon concentrations remained fairly stable during the 72-hour test period with average Carbon concentrations at levels corresponding to 30-48% relative to the initial loading rates. Since TOC-analysis is a non-specific method, no further estimations for exposure concentrations were performed. The results were consequently reported in terms of loading rates and expressed in mg per litre. In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, Soybean oil, epoxidized, Me ester, reaction products with propylene glycol reduced growth rate of this fresh water algae species significantly at loading rates of 32 mg/La EL50nd higher. The growth rate inhibition (72h-ERL50) exceeded the highest loading rate tested of 100 mg/L. The72h-ERL10 and 72h-NOELR for growth rate inhibition was 39 mg/L (95% Cl 30 - 45 mg/L) and 18 mg/L respectively based on loading rates. While 72h-EYL50, 72h-ERL10 and 72h-NOELR for yield inhibition was 52 mg/L (95% Cl 47 - 59 mg/L), 14 mg/L (95% Cl 10 -17 mg/L) and below 10 mg/L respectively based on loading rates.

 

 

Description of key information

A full OECDTG 201 GLP test was performed with Pseudokirchneriella subcapitata. Six exponentially growing algal cultures were exposed for 72h to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rates of10, 18, 32, 56 and 100 mg/L of Soybean oil, epoxidized, Me ester, reaction products with propylene glycol.The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for Total Organic Carbon (TOC) analysis were taken at the start and at the end of exposure.Measured Carbon concentrations remained fairly stable during the 72-hour test period with average Carbon concentrations at levels corresponding to 30-48% relative to the initial loading rates. Since TOC-analysis is a non-specific method, no further estimations for exposure concentrations were performed. The results were consequently reported in terms of loading rates and expressed in mg per litre. In conclusion, under the conditions of the present study withPseudokirchneriella subcapitata, Soybean oil, epoxidized, Me ester, reaction products with propylene glycol reduced growth rate of this fresh water algae species significantly at loading rates of 32 mg/LaEL50nd higher. The growth rate inhibition (72h-ERL50) exceeded the highest loading rate tested of 100 mg/L. The72h-ERL10 and 72h-NOELR for growth rate inhibition was 39 mg/L (95% Cl 30 - 45 mg/L) and 18 mg/L respectively based on loading rates. While 72h-EYL50, 72h-ERL10 and 72h-NOELR for yield inhibition was 52 mg/L (95% Cl 47 - 59 mg/L), 14 mg/L (95% Cl 10 -17 mg/L) and below 10 mg/L respectively based on loading rates.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
39 mg/L

Additional information

The ErL50 value is > 100 mg/L. The ErL50 and ErL10 values are based on loading.