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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 March 2017 - 20 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
28 July 2011.
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: EM14089719
- Expiration date of the lot/batch: 01 August 2017
- Purity test date: 07/07/2016 ( certificate of analysis)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle:
Solubility in vehicle:
• Water 647.0 mg/L, at 20°C and pH= 6.3 (OECD 105)
• Other: …… Not indicated
Stability in vehicle:
• Water Not available
• Other: ….. Not indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: n/a
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: 10, 18, 32, 56 and 100 mg/L
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS:
Test facility test item number: 207812/A
Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Octan-3-one
Volatile: Epiwin prediction vp of ~300 Pa
Specific gravity/density: Not available

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Single samples for possible analysis were taken from all test concentrations and the control
according to the schedule below.

- Sampling method:
Frequency: t=0 h, t=24 h and t=72 h
Volume: 4.0 mL
At the end of the exposure period, the replicates with algae were not 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

- Sample storage conditions before analysis:
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility. Additionally, reserve samples of 4.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method:
Preparation of test solutions started with the highest concentration of 100 mg/L applying a magnetic stirring period of 47 minutes (combined limit/range-finding test) or 58 minutes(final test) to accelerate the dissolving of the test item. Lower test concentrations wereprepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

After preparation, volumes of 120 mL (combined limit/range-finding) or 40 mL (final test)
were added to each replicate of the respective test concentration. Subsequently, 2.4 mL
(combined limit/range-finding) or 0.8 mL (final test) of an algal suspension were added to
each replicate providing a cell density of 104 cells/mL.

- Controls: Test medium without test item or other additives
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none

Test organisms

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): pre-culture medium and conditions are the same

Study design

Test type:
static
Water media type:
freshwater
Remarks:
closed system
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
22.3 - 23.1°C
pH:
t=0h: 7.4
t = 72h: 7.6- 8.3
Nominal and measured concentrations:
nominal concentrations: 4.6, 10, 22, 46 and 100 mg/L

Measured concentrations: 3.2, 6.9, 17, 36 and 82 mg/L ( Time Weighted Average (TWA) exposure concentrations)

Nominal concentration of ethyl amyl ketone(mg/L): Measured concentration t=0h, t=24h, t=72h, TWA (mg/L)
4.6mg/L: 3.54, 3.04,3.19,3.2
10 mg/L: 6.04, 6.99, 7.16, 6.9
22 mg/L: 16, 17.6, 16.7, 17
22 mg/L (without algae.): 19.6, 18.5, 19.3, 19
46 mg/L: 37.6, 38.1, 33.2, 36
100 mg/L: 84.0, 86.7, 75.8, 82
Details on test conditions:
TEST SYSTEM
- Test vessel: 40 mL, purge & trap vials, containing 40 mL of test solution.
- Type: Closed, no headspace.
- Aeration: none
- Initial cells density: 1x 10^4 cells/mL
- Control end cells density: 264.7 x10^4 cells/mL)
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates): 6
- No. of vessels per control (no algae): 2
- No of extra vessels: 1 or 2 extra replicate of each test group for sampling after 24 hours

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:
M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

TEST MEDIUM / WATER PARAMETERS
Adjusted M2; according to the OECD 201 Guideline, 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 300 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
HEPES buffer 6 mmol/L
pH 7.1 ± 0.3

- Culture medium different from test medium: yes
- 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.

OTHER TEST CONDITIONS
Illumination: TLD-lamps with a light intensity within the range of 90 to 92 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.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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 cuvettes (path length =10 mm). Algal medium was
used as blank

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study
- Test concentrations:0.10,1,10,100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on statistical analysis
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
36 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on biological relevance
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
38 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% cl (37-38)
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
50 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% cl (49-51)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 82 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Based on statistical significance
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.9 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: Based on biological relevance
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
20 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% cl (20-21)
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
26 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% cl (25-27)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
41 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% cl (40-42)
Details on results:
- Exponential growth in the control (for algal test): yes N=266 (> 16)
- The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was: 0.5% (, 7%)
- The mean of the replicate coefficients of variation for section-by-section growth rate in the control was: 16.7% (< 35%)

- Observation of abnormalities (for algal test): No. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 36 and 82 mg/L when compared to the control. Other concentration levels were not assessed.

There were a couple deviations including a different test vessel being used instead of 120 mL glass Erlenmeyer vessels and not shaking the test vessels for both control and treatments during the final test for one night. However all validity criteria for the control were met and therefore this had no effect on the outcome/validity of the test.
Results with reference substance (positive control):
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 NOEC and the EC50 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 EC10 and EC20 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 ECx 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 average exposure concentrations of the test item. No EC50-values could be calculated for growth rate (EC50 > maximum concentration tested). The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Growth Rate (day-1) and Percentage Inhibition for the Total Test Period

TWA concentration of

Octan-3-one (ethyl amyl

ketone) (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.861

0.0091

6

 

3.2

1.854

0.0098

3

0.4

6.9

1.850

0.0077

3

0.6*/#

17

1.823

0.0025

3

2.0*/#

36

1.703

0.0032

3

8.5*/#

82

0.978

0.0098

3

47.5*

 

* - effect was statistically significant

#- effect biologically irrelevant (<10%)

Yield (x104cells/mL) and Percentage Inhibition for the Total Test Period

TWA concentration of

Octan-3-one (ethyl amyl

ketone) (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

264.7

7.30

6

 

3.2

259.2

7.62

3

2.1

6.9

256.0

5.88

3

3.3*/#

17

236.2

1.80

3

10.8*

36

164.4

1.57

3

37.9*

82

17.8

0.56

3

93.3*

 

* - effect was statistically significant

#- effect biologically irrelevant (<10%)

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Pseudokirchneriella subcapitata, Octan-3-one (ethyl amyl ketone) reduced growth rate of this fresh water algae species statistically significantly at TWA concentrations of 6.9 mg/L and higher. The EC50 for growth rate inhibition (72h-ERC50) exceeded 82 mg/L, being the highest tested TWA concentration. The EC50 for yield inhibition (72h-EYC50) based on TWA concentrations was 41 mg/L with a 95% confidence interval ranging from 40 to 42 mg/L. The EC10 for growth rate inhibition (72h-ERC10) based on TWA concentrations was 38 mg/L with a 95% confidence interval ranging from 37 to 38 mg/L. The EC10 for yield inhibition (72h-EYC10) based on TWA concentrations was 20 mg/L with a 95% confidence interval ranging from 20 to 21 mg/L. The 72h-NOEC for both growth rate inhibition and yield inhibition was 3.2 mg/L based on statistical significance. The 72h-NOEC for growth rate was 36 mg/L based on biological significance.
Executive summary:

In this study Octan-3-one (ethyl amyl ketone) was tested for its ability to generate toxic effects in Pseudokirchneriella subcapitata during an exposure period of 72 hours. The study was based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011.

Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominal Octan-3-one (ethyl amyl ketone) concentrations of 4.6, 10, 22, 46 and 100 mg/L in closed systems. At the start of the test, the actual measured test concentrations were at 60 to 89% relative to nominal. During the 72-hour test period concentrations remained stable or decreased slightly. Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated to correspond with 3.2, 6.9, 17, 36 and 82 mg/L for the nominal concentrations of 4.6, 10, 22, 46 and 100 mg/L, respectively. The substance reduced growth rate statistically significantly at TWA concentrations of 6.9 mg/L and higher. The 72h-NOEC for both growth rate inhibition and yield inhibition was 3.2 mg/L based on statistical significance. The 72h NOEC for growth rate based on biological significance was 36 mg/L . Based on TWA exposure concentrations the 72h-ErC10, 72h-EYC10, 72h-EYC50and 72h-ErC50 were found to be 38 mg/L (95%Cl 37 -38), 20mg/L (95%Cl 20 -21mg/L) ,41 mg/L(95%Cl 40-42mg/L) and> 82 mg/L respectively