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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:
3 Oct 2017 - 6 Oct 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:
yes
Remarks:
Path length of the immersion probe used to measure cell density was 10 mm (this has no influence on the results).
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
During the test, 4.0 mL samples were taken for possible analysis from the test concentration and controls. Sampling frequency was at the start of the test (0 hours), at 24 hours and at the end of the test (72 hours). Samples were analysed on the day of sampling. At the end of the exposure period, the replicates were pooled at each concentration before sampling. Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Vehicle:
no
Details on test solutions:
Water solubility of the test item was determined in Test Facility Study no. 517600 and was 2.0 ¿g/L at a loading of 10 mg/L and 10 ¿g/L at a loading rate of 500 mg/L. As determined in 517599, the test item is slightly surface active, potentially causing an overestimation of the water solubility.

The batch of Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol tested was completely soluble in test medium at the limit test concentration tested. No correction was made for the purity/composition of the test item.

Preparation of the test solution started with a stock solution of 0.10 mg/mL in acetone. No other treatment than vigorous shaking was needed to completely dissolve the test item. Thereafter, 100 ¿L of the stock was spiked into 1 litre of medium to reach a test item concentration of 10 ¿g/L and a 15-minute period of magnetic stirring was applied to ensure homogenous distribution. All test solutions were clear and colourless at the end of the preparation procedure.

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.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test species source: In-house laboratory culture (Charles River Laboratories Den Bosch BV Hambakenwetering 7 5231 DD ¿s-Hertogenbosch The Netherlands).
Reason for selection: The test organism is an unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

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 immersion probe (pathlength =10 mm). Algal medium was used as blank.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
not specified
Test temperature:
22 - 23°C
pH:
7.9 - 8.0
Dissolved oxygen:
NA
Salinity:
NA
Conductivity:
NA
Nominal and measured concentrations:
Test algal cultures were exposed to a blank control, a solvent control and a limit concentration of test item, with 6 replicates of each. One extra replicate of each group was included for sampling purposes at 24 hour of exposure two replicates of the limit concentration without algae.

Analysis of test item samples at the start of the test showed a measured concentration comparable to nominal (9.7 ¿g/L). The measured concentration decreased to 0.68 and 0.40 ¿g/L (7.0 and 4.1% of initial) after 24 and 72 hours of exposure, respectively. Based on these results, the Time Weight Average (TWA) exposure concentration was 1.2 ¿g/L. Concentrations measured in the solution incubated without algae showed similar results. This, and the fact that undissolved material was observed in the vessel incubated without algae at the end of the exposure period, indicates that the observed decrease was not due to the growing algal biomass but rather solubility of the test item in the medium. Therefore, the TWA concentration of 1.2 ¿g/L was considered the highest soluble in test medium.
Details on test conditions:
Test vessels: 100 mL, all-glass, containing 50 mL of test solution.
Test 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 79 to 86 ¿E.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.

Appearance of the algal cells were measured at the end of the final test by performing microscopic observations on the concentration tested to observe for any abnormal appearance of the algae. Measurements of pH were conducted at the beginning and at the end of the test for the test. Temperature of the test medium was measured continuously in a temperature control vessel.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (Concentrations: 0.18, 0.32, 0.56, 1.0, 1.8, 3.2 mg/L and a control)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.2 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 1.2 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 1.2 µg/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.:
> 1.2 µg/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.:
1.2 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 1.2 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 1.2 µg/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.:
> 1.2 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
Analysis of the samples taken at the start of the test showed a measured concentration comparable to nominal (9.7 ¿g/L). The measured concentration decreased to 0.68 and 0.40 ¿g/L (7.0 and 4.1% of initial) after 24 and 72 hours of exposure, respectively. Based on these results, the Time Weight Average (TWA) concentrations were calculated. Concentrations measured in the solution incubated without algae showed similar results. This, and the fact that undissolved material was observed in the vessel incubated without algae at the end of the exposure period, indicates that the observed decrease was not due to the growing algal biomass but rather solubility of the test item in the medium. Therefore, the TWA concentration of 1.2 ¿g/L was considered the highest soluble in test medium.

A stimulation, rather than inhibition of both algal growth rate and yield was observed in the study. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the test concentration when compared to the controls.
Results with reference substance (positive control):
Exposure of Pseudokirchneriella subcapitata to the reference substance (potassium dichromate) at concentrations of 0.18, 0.32, 0.56, 1.0, 1.8, 3.2 mg/L and to a control for a maximum of 72 hours showed responses within the ranges of the expected responses at the different concentrations. Potassium dichromate inhibited growth rate of P. subcapitata at nominal concentrations of 0.32 mg/L. The 72h-ErC50 for growth rate was 1.1 mg/L with a 95% confidence interval ranging from 1.1 to 1.1 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The 72h-EyC50 for yield was 0.36 mg/L with a 95% confidence interval ranging from 0.35 to 0.36 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L.

Growth rate and percentage inhibition for the total test period

Test item1

TWA concentration (¿g/L)

Mean growth rate

Std. Dev.

n

% Inhibition

Pooled Control

1.742

0.0325

12

 

1.2

1.751

0.0201

6

-0.5

1 Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol

 

Growth rate and percentage inhibition at different time intervals

Test item 1

TWA concentration (¿g/L)

n

0 ¿ 24 h

24 - 48 h

48 ¿ 72 h

Mean

% inhibition

Mean

% inhibition

Mean

% inhibition

Pooled Control

6

1.451

 

1.950

 

1.826

 

1.2

6

1.490

-2.7

1.883

3.4

1.879

-2.9

1 Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol

 

 

Yield and percentage inhibition for the total test period

Test item1

TWA concentration (¿g/L)

Mean growth rate

Std. Dev.

n

% Inhibition

Pooled Control

186.0

17.67

12

 

1.2

190.4

11.73

6

-2.4

1 Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2-dicarboxylic anhydride and propylidenetrimethanol

Validity criteria fulfilled:
yes
Remarks:
The study met the acceptability criteria prescribed by the study plan and was considered valid.
Conclusions:
No inhibition of growth rate or inhibition of yield was recorded in Pseudokirchneriella subcapitata under the test conditions at the concentration of 1.2 ¿g/L (considered the highest soluble concentration in test medium). The EC10 and EC50 for growth rate inhibition (72h-ERC10 and 72h-ERC50) and yield inhibition (72h-EYC10 and 72h-EYC50) were beyond the range tested, i.e. exceeded the TWA concentration of 1.2 ¿g/L. The 72h-NOEC for both growth rate and yield inhibition was 1.2 ¿g/L.
Executive summary:

A study investigating the toxicity of Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2 -dicarboxylic anhydride and propylidenetrimethanol to the freshwater algae species, Pseudokirchneriella subcapitata, was conducted according to the OECD Guideline No. 201 at a GLP accredited laboratory. The study met the acceptability criteria prescribed by the study plant and was considered valid.

Six exponentially growing algal cultures per group, were exposed to a blank control, a solvent control and to a limit concentration, i.e. 10 ¿g/L. 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 of the samples taken at the start of the test shows a measured concentration comparable to nominal (9.7 µg/L). The measured concentration decreased to 0.68 and 0.40 µg/L (7.0 and 4.1% of initial) after 24 and 72 hours of exposure, respectively. The calculated average exposure concentrations was 1.2 µg/L.

No significant inhibition for both growth rate and yield was observed at the limit concentration tested, therefore the EC50 values for growth rate (ErC50) and yield (EyC50) were both >1.2 µg /L and the NOEC values for growth rate and yield were both 1.2 µg/L.

Description of key information

A study investigating the toxicity of Epoxypropyl neodecanoate, oligomeric reaction products with cyclohexane-1,2 -dicarboxylic anhydride and propylidenetrimethanol to the freshwater algae species, Pseudokirchneriella subcapitata, was conducted according to the OECD Guideline No. 201 at a GLP accredited laboratory. The study met the acceptability criteria prescribed by the study plan and was considered valid.

Six exponentially growing algal cultures per group, were exposed to a blank control, a solvent control and to a limit concentration, i.e. 10 ¿g/L. 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 of the samples taken at the start of the test showed a measured concentration comparable to nominal (9.7 µg/L). The measured concentration decreased to 0.68 and 0.40 µg/L (7.0 and 4.1% of initial) after 24 and 72 hours of exposure, respectively. The calculated average exposure concentrations was 1.2 µg/L.

No significant inhibition for both growth rate and yield was observed at the limit concentration tested, therefore the EC50 values for growth rate (ErC50) and yield (EyC50) were both >1.2 µg /L and the NOEC values for growth rate and yield were both 1.2 µg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
1.2 µg/L
EC10 or NOEC for freshwater algae:
1.2 µg/L

Additional information