3-Pentanone, 1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-, reaction products with 2-propyn-1-ol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The test was conducted between 12 June 2016 and 20 August 2016

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)

Version / remarks:

28 July 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

07 December 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Version / remarks:

December 14, 2000

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Solubility in water Predicted to be between 1 and 10 mg/L

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from all test concentrations and the control at t=0 h, t=24 h and t=72 h.

Vehicle:

no

Details on test solutions:

The batch of Grisalva tested was a clear yellow liquid mixture and not completely soluble in test medium at the concentrations tested. Test solutions were prepared separately at loading rates ranging from 0.16 to 100 mg/L, applying a 24-hours period of magnetic stirring after which the resulting mixtures were allowed to stabilize for 1 hour. Subsequently, the Water Accommodated Fractions (WAFs) were siphoned off over glass wool. All final test solutions were clear and colorless.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
The test was carried out using an in-house laboratory culture of Pseudokirchneriella subcapitata, strain NIVA CHL 1. Algae stock cultures were started by inoculating growth medium (M1 medium) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (60 to 120 μE/m2/s) in a climate room at a temperature of 21-24°C.

PRE-CULTURE
3 days before the start of the test, cells from the algal stock culture were inoculated in M2 culture medium (same medium used for the range-finding and definitive tests) at a cell density of 10000 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Hardness M2 medium (Ca+Mg): 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

Temperature of medium was measured continuously in a temperature control vessel and was maintained at 21-23°C throughout the test.

pH:

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was maintained within 7.1 - 7.9.

Nominal and measured concentrations:

Based on the results of the range-finding test the following test concentrations were assigned to the definitive test:
First full test: Nominal concentrations: WAFs prepared at loading rates of 10, 18, 32, 56 and 100 mg/L
Second full test: Nominal concentrations: WAFs prepared at loading rates of 0.16, 0.80, 4.0, 20 and 100 mg/L.
The second full test was performed with a concentration spacing factor of 5.0. The preceding test (first full test) with a spacing factor of 1.8 resulted in a flat response curve which did not allow for the determination of a NOEC.

The measured concentrations in the first full test were determined at nominal concentrations of 10 and 100 mg/L, respectively, and found to be 1.27 and 1.55 mg/L, decreasing to 78% and 70% of initial after 24 hours and to 9.1-18% (duplicate vessels) and 22% after 72 hours.

In the second full test (final test), samples taken from the control and all test concentrations were analyzed. The initial test item concentrations were 0.033, 0.091, 0.60, 0.74 and 3.6 mg/L in WAFs prepared at 0.16, 0.80, 4.0, 20 and 100 mg/L, respectively. Measured concentrations were at the level of 59-116% of initial after 24 hours of exposure and at 10-40% at 4.0-100 mg/L nominal, and below the level of detection (<0.033 mg/L) at 0.16 and 0.80 mg/L nominal, at the end of the test.The test item was not detectable in medium from control replicates.
Based on these results, the Time Weight Average (TWA) concentrations were calculated to be 0.019, 0.048, 0.31, 0.45 and 2.1 mg/L in WAFs prepared at loading rates of 0.16, 0.80, 4.0, 20 and 100 mg/L. The concentration in the WAF of 4.0 mg/L incubated without algae decreased less than in the solution incubated with algae.

The EC50 values were expressed in terms of the average (geometric mean) exposure concentrations.

The measured concentrations in the final test are shown in a Table included under "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel:
120 mL, all-glass, containing 120 mL of test solution, with minimum headspace and closed airtight were used for the control and each treatment group.

The control group was maintained under identical conditions but not exposed to the test material.
- Initial cells density: After preparation of the test solutions, volumes of 120 mL were added to each replicate of the respective test concentration containing 2.4 mL of an algal suspension, providing a cell density of 10000 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None (not applicable)

GROWTH MEDIUM
- Standard medium used: no
Adjusted M2; Standard M2 according to the OECD 201 Guideline but with a larger amount of NaHCO3, and the addition of HEPES buffer to reduce pH changes. This medium was formulated using Milli-RO water and will have 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
HEPES buffer 6 mmol/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 7.1 ± 0.3
- Photoperiod: Continuous light using TLD-lamps with a light intensity within the range of 68 to 78 μE.m-2.s-1. Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Salinity: Not applicable
- Determination of cell concentrations: counting and spectrophotometer
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined. 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 and the extra replicates as background for the treated solutions. Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.
- Results used to determine the conditions for the definitive study:
A combined limit/range-finding study was conducted at nominal concentrations of 0 (control) and WAFs prepared at 1.0, 10 and 100 mg/L. Six replicates of exponentially growing algae were exposed to a control and a WAF prepared at 100 mg/L. Three replicates per concentration were exposed to WAFs prepared at 1.0 and 10 mg/L. Cell densities were recorded at 24-hour intervals in the control and the highest concentration. Intermediate concentrations were measured only at the end of the exposure period. At 1.0, 10 and 100 mg/L, respectively, the percent inhibition of growth rate was 7.75, 11.6 and 6.3%, and the percent inhibition of yield was 31.9, 43.8 and 26.4%. The range of loading rates to be tested in the first full test was chosen based on an assumption that the effect observed in WAF prepared at 10 mg/L was an artefact, rather real effect.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (tested between 18 and 21 July 2016)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.53 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Please refer to the tables under "Any other information on results incl. tables".

Results with reference substance (positive control):

The EC50 for growth rate inhibition (72h-ERC50) was 1.0 mg/L with a 95% confidence interval ranging from 0.97 to 1.0 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 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 0.20 and 1.1 mg/L. The observed 72h-EYC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

The p(Chi2) value for the overall fit for growth rate was 1.00000. The p(F) value for the slope of the regression curve for growth rate was 0.00400. For confidence intervals of EC-values please refer to the table with effect parameters under "Any other information on results incl. tables".

Detailed results on percentage inhibition of growth rate and all effect parameters for growth rate are presented in the tables below.

Table 1: Percentage inhibition of growth rate (total test period) during the final test

Grisalva TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.643	0.0174	6
0.019	1.631	0.0191	3	0.7
0.048	1.635	0.0202	3	0.5
0.31	1.498	0.0112	3	8.8*^#
0.45	1.521	0.0076	3	7.4*^#
2.1	1.271	0.0386	3	22.7*

* - effect was statistically significant

^#- effect biologically insignificant (<10%)

Table 2: Effect parameters for growth rate

	Parameter (mg/L)	NOEC	EC10	EC20	EC50
Growth rate	Value	0.45	0.53	1.7	>2.1
	lower 95%-cl		0.27	1.2	n.d.
	upper 95%-cl		0.77	2.7	n.d.
Yield	Value	0.048	0.076	0.18	0.89
	lower95%-cl		0.056	0.14	0.78
	upper 95%-cl		0.10	0.21	1.0

n.d. – not determined

Table 3: Concentrations of the test item in test medium – final test 2

Time of sampling [hours]	Loading rate^1 [mg/L]	Concentration analysed [mg/L]	Relative to initial [%]
0	0	n.d.
	0.16	0.033^3
	0.80	0.091
	4.0	0.599
	4.0^2	0.629
	20	0.737
	100	3.56
24	0	n.d.	n.a.
	0.16	<LOD^4	n.a.
	0.80	0.067^3	74
	4.0	0.535	89
	4.0^2	0.646	103
	20	0.855	116
	100	2.11	59
72	0	n.d.	n.a.
	0.16	<LOD^4	n.a.
	0.80	<LOD^4	n.a.
	4.0	0.060^3	10
	4.0^2	0.451	72
	20	0.0952	13
	100	1.41	40

¹    A water accommodated fraction (WAF) prepared at the loading rate.

²    Without algae.

³    Estimated value, calculated by extrapolation of the calibration curve.

⁴    The limit of detection of the method was determined to be 0.033 mg/L taking a dilution factor of 0.125 into account.This concentration was based on the result of the sample with loading rate 0.16 mg/l at t= 0 hours. All 4 quantifiable peaks were visible and highenough for adequate detection and estimated quantitation. The chromatogram of a lower concentration than 0.033 mg/L would only show 3 out of 4 peaks, therefore the test item could not be considered as detected.

n.d.    Not detected.

n.a. Not applicable.

Validity criteria fulfilled:

yes

Remarks:

In the control, cell density increased by an average factor of at least 16 (i.e. 138). The mean CV for section-by-section specific growth rates was <35% (i.e. 20%). The CV of average specific growth rates during the whole test period was <7% (i.e. 1.1%).

Conclusions:

The ErC50, ErC10 and NOEC were >2.1, 0.53 and 0.45 mg/L, respectively.

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201 and was conducted under GLP.The substance was a clear yellow liquid mixture. In the preliminary test the substance was not completely soluble in test medium at the concentrations tested. Due to the low solubility and complex nature of the test item, the test solutions were prepared as Water Accommodated Fractions (WAFs), in line with OECD Guidance 23 on difficult substances (2000). Following apreceding combined limit/range-finding and first full test,six exponentially growing algal cultures (starting with 10000 cells/ml) were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at 0.16, 0.80, 4.0, 20 and 100 mg/L. Samples for analytical confirmation of test item concentrations were taken at the start, after 24 hours of exposure and at the end of the test. The exposure was performed in airtight closed vessels with headspace reduced to minimum. The initial test item concentrations were 0.033, 0.091, 0.60, 0.74 and 3.6 mg/L in WAFs prepared at 0.16, 0.80, 4.0, 20 and 100 mg/L, respectively. Measured concentrations were at the level of 59-116% of initial after 24 hours of exposure and at 10-40% at 4.0-100 mg/L nominal, and below the level of detection (<0.033 mg/L) at 0.16 and 0.80 mg/L nominal, at the end of the test. Based on these results, the Time Weight Average (TWA) concentrations were calculated to be 0.019, 0.048, 0.31, 0.45 and 2.1 mg/L, respectively, and used to determine the effect parameters. The study met the acceptability criteria prescribed by the study plan and was considered valid. The effect parameters obtained in this study were:EC50 (72h): >2.1 mg/L test mat. (meas. (geom. mean)) based on: growth rate; NOEC (72h): 0.45 mg/L test mat. (meas. (geom. mean)) based on: growth rate; EC10 (72h): 0.53 mg/L test mat. (meas. (geom. mean)) based on: growth rate.

Description of key information

Acute and long-term algae toxicity according to OECD TG 201: ErC50 and EC10 are > 2.1 and 0.53 mg/l, respectively.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.53 mg/L

Additional information

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201 and was conducted under GLP. The substance was a clear yellow liquid mixture. In the preliminary test the substance was not completely soluble in test medium at the concentrations tested. Due to the low solubility and complex nature of the test item, the test solutions were prepared as Water Accommodated Fractions (WAFs), in line with OECD Guidance 23 on difficult substances (2000). Following apreceding combined limit/range-finding and first full test, six exponentially growing algal cultures (starting with 10000 cells/ml) were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at 0.16, 0.80, 4.0, 20 and 100 mg/L. Samples for analytical confirmation of test item concentrations were taken at the start, after 24 hours of exposure and at the end of the test. The exposure was performed in airtight closed vessels with headspace reduced to minimum. The initial test item concentrations were 0.033, 0.091, 0.60, 0.74 and 3.6 mg/L in WAFs prepared at 0.16, 0.80, 4.0, 20 and 100 mg/L, respectively. Measured concentrations were at the level of 59-116% of initial after 24 hours of exposure and at 10-40% at 4.0-100 mg/L nominal, and below the level of detection (<0.033 mg/L) at 0.16 and 0.80 mg/L nominal, at the end of the test. Based on these results, the Time Weight Average (TWA) concentrations were calculated to be 0.019, 0.048, 0.31, 0.45 and 2.1 mg/L, respectively, and used to determine the effect parameters. The study met the acceptability criteria prescribed by the study plan and was considered valid. The effect parameters obtained in this study were: EC50 (72h): >2.1 mg/L test mat. (meas. (geom. mean)) based on: growth rate; NOEC (72h): 0.45 mg/L test mat. (meas. (geom. mean)) based on: growth rate; EC10 (72h): 0.53 mg/L test mat. (meas. (geom. mean)) based on: growth rate.