Reaction mass of 1-(2,6,6-trimethylcyclohex-2-en-1-yl)hepta-1,6-dien-3-one and 1-(2,6,6-trimethylcyclohex-1-en-1-yl)hepta-1,6-dien-3-one

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from a gudieline study

Justification for type of information:

The algae toxicity of Hexalon is based on read across from Galbascone. The documentation is presented in the Endpoint summary of Aquatic toxicity.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.3 mg/L

Basis for effect:

growth rate

Remarks on result:

other: read-across from Galbascone

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2 mg/L

Basis for effect:

growth rate

Remarks on result:

other: read-across from Galbascone

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.1 mg/L

Basis for effect:

growth rate

Remarks on result:

other: read-across from Galbascone

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 August - 05 December 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information is used to read across to Hexalon.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(2006; Annex 5 corrected 28 July 2011)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

(2008, amended in 2009)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test concentrations and the control according to the schedule below.

Frequency: at t=0, 24 and 72 h
Volume: 3.0 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis

At the end of the exposure period, samples were taken from one replicate of each test concentration.

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:

PREPARATION AND APPLICATION OF TEST SOLUTION
The batch of GALBASCONE tested was a clear colourless liquid and not completely soluble in test medium at loading rates above 0.6 mg/L. No correction was made for the composition of the test item.

All test solutions were prepared separately applying one day of magnetic stirring in a closed vessel followed by a stabilization period of one day in order to reach the maximum solubility of the test item in the test medium. The resulting clear and colourless solutions (concentrations up to and including 0.6 mg/L) and the aqueous mixtures (concentrations above 0.6 mg/L) were siphoned off. The final test solutions were all referred to as Water Accommodated Fractions (WAFs).

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: in-house laboratory culture

ACCLIMATION
- Pre-culture:
3 to 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (adjusted M2) 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.

After preparation, volumes of appr. 120 mL test solution were added to each replicate of the respective test concentration. Subsequently, 2.4 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL. The total fill volume of each vessel was appr. 120 mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

Temperature was maintained between 21 and 23°C throughout the test.

pH:

t=0: 7.4
t=72 h: 7.5-8.0

Nominal and measured concentrations:

Combined limt/range-finding test - WAF loading rates: 1.0, 10 and 100 mg/L.

Final test:
Based on the results of the range-finding test the following WAF loading rates were assigned to the final test: 0.2, 0.6, 2.0, 6.0, 20 and 60 mg/L.
Time-weighted average (TWA) test concentrations: 0.14, 0.43, 1.3, 3.6, 15 and 34 mg/L, see table 1 in field "any other information on results' for more details.

The effect concentrations are based on TWA concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 120 mL all-glass, air-tight closed vessels with minimal headspace to prevent loss of test substance due to volatilisation; fill volume: approx. 120 mL .
The control group was maintained under identical conditions but not exposed to the test material.
- Initial cells density: 1 x 10^4 cells per mL
- No. of vessels per concentration (replicates): 3 + 1 extra replicate of each test concentration for sampling purposes
- No. of vessels per control (replicates): 6 +1 extra replicate of the control for sampling purposes
- No. of vessels without algae (replicates): 2 (of the WAF at loading rate of 2.0 mg/L)

GROWTH MEDIUM
- Stock culture medium: M1 prepared according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Pre-culture and test medium: adjusted M2 (prepared in accordance with OECD 201 using reverse osmosis purified deionised water (Milli-RO, Millipore); larger amount of NaHCO3, addition of HEPES buffer and a lower pH - pH adjusted to 7.1 ± 0.3)

- llumination: continuously using TLD-lamps with a light intensity within the range of 77 to 87 µE.m-2.s-1 while constantly shaking.

- Determination of cell concentrations: Cells were counted using a microscope and a counting chamber.

- Appearance of the cells: At the end of the final test microscopic observations were performed on the 6.0 mg/L WAF to observe for any abnormal appearance of the algae.

- Results used to determine the conditions for the definitive study:
The results of the combined limit/range-finding test showed that expected EC50 for growth rate inhibition was slightly above the concentration obtained in a WAF prepared at a loading rate of 10 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (September 2015)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.5 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 6.0 - 7.1 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 2.1- 2.8 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities in algal cells: no

Statistically significant inhibition of growth rate was found at TWA measured concentrations of 1.3 mg/L and higher. Biologically relevant inhibition of growth rate (>10%) was found at TWA measured concentrations of 3.6 mg/L and higher. The NOEC is set at 1.3 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72h-ErC50: 1.2 mg/L (95% confidence interval ranging from 1.1 to 1.2 mg/L)
- Other: 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.

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 (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller) or significant inhibition of yield (Welch-t test for Inhomogeneous Variances with Bonferroni-Holm Adjustment, α=0.05, one-sided, smaller).

Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the ccorresponding TWA concentrations of the test substance.

The calculations were performed with ToxRat Professional v. 3.0.0 (ToxRat Solutions® GmbH, Germany).

Measured test substance concentrations - final test

At the start of the test, the measured test concentrations were 0.17, 0.52, 1.6, 4.6, 18 and 39 mg/L in the WAFs prepared at loading rates of 0.2, 0.6, 2.0, 6.0, 20 and 60 mg/L, respectively (65 -88% of loading rate, calculated by reviewer). These concentrations remained stable during the first 24 hours of exposure (82-96% of initial) but decreased to 53-75% of initial at the end of the test period. The measured concentration in the sample taken from the 2.0 mg/L concentration without algae also decreased but remained higher (71% of initial after 72 hours of exposure) then in the vessel with algae (53% of initial after 72 hours of exposure). Given this result, the TWA concentrations were calculated and used to determine the effect parameters.

Table 1: Concentrations of the test item in test medium - final test

Time of sampling [hours]	Loading rate^1 [mg/L]	Concentration analysed [mg/L]	Relative to initial [%]
0	0	n.d.
	0.2	0.166
	0.6	0.518
	2.0	1.64
	2.0^2	1.68
	6.0	4.64
	20	17.6
	60	39.2
24	0	n.d.
	0.2	0.160	96
	0.6	0.461	89
	2.0	1.53	94
	2.0^2	1.60	95
	6.0	3.79	82
	20	15.7	89
	60	34.9	89
72	0	n.d.
	0.2	0.114	69
	0.6	0.337	65
	2.0	0.860	53
	2.0^2	1.20	71
	6.0	2.99	65
	20	12.7	72
	60	29.5	75

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

²          Without algae.

n.d.    Not detected.

n.a.     Not applicable.

Table 2: Measured concentrations versus WAFs prepared at various loading rates

GALBASCONE WAF prepared at x mg/L	Measured concentration (mg/L)			TWA conc. (mg/L)
	t=0h	t=24h	t=72 h
0.2	0.166	0.160	0.114	0.14
0.6	0.518	0.461	0.337	0.43
2.0	1.64	1.53	0.860	1.3
2.0^1	1.68	1.60	1.20	1.5
6.0	4.64	3.79	2.99	3.6
20	17.6	15.7	12.7	15
60	39.2	34.9	29.5	34

¹Without algae

Inhibition results - final test

Table 3: Percentage inhibition of growth rate (total test period) during the final test

TWA conc. GALBASCONE (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.587	0.0374	6
0.14	1.595	0.0237	3	-0.5
0.43	1.590	0.0198	3	-0.2
1.3	1.467	0.0251	3	7.6^#
3.6	1.264	0.0145	3	20.4*
15	0.199	0.1319	3	87.4*
34	0.045	0.0780	3	97.2*

* - effect was statistically significant

^#- effect was statistically significant but not considered biologically relevant (<10%)

Validity criteria fulfilled:

yes

Remarks:

In controls: cell density increased by an average factor of >16 within 72 hours, mean CV for section-by-section specific growth rates only slightly exceeded 35% and CV of average specific growth rates during the whole test period did not exceed 7%

Conclusions:

The ErC50, ErC10 and NOEC were 6.5, 2.4 and 1.3mg/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 GLP. Water Accommodated Fractions (WAFs) were separately prepared in closed vessels at loading rates of 0.2, 0.6, 2.0, 6.0, 20 and 60 mg/L applying a 1 -day period of magnetic stirring in closed vessels followed by a 1 -day stabilisation period. Up to 0.6 mg/L the substance was soluble in test medium, higher concentrations formed mixtures, which were siphoned off and used as test solutions. The final test solutions were all referred to as Water Accommodated Fractions (WAFs). Pseudokirchneriella subcapitata was exposed for 72 hours (three replicate flasks per concentration, six replicate flasks per control) under constant illumination and shaking at a temperature of 21 - 23°C.

Samples were taken from all treatments at t = 0 , 24 and 72 h and analysed with a validated GC-FID method. At the start of the test, the measured test concentrations were 0.17, 0.52, 1.6, 4.6, 18 and 39 mg/L in the WAFs prepared at loading rates of 0.2, 0.6, 2.0, 6.0, 20 and 60 mg/L, respectively (65 -88% of loading rate). These concentrations remained stable during the first 24 hours of exposure (82-96% of initial) but decreased to 53-75% of initial at the end of the test period. Therefore the TWA measured concentrations were calculated and used for determination of effect concentrations. Loading rates of 0.2, 0.6, 2.0, 6.0, 20 and 60 mg/L corresponded with TWA measured concentrations of 0.14, 0.43, 1.3, 3.6, 15 and 34 mg/L , respectively.

Statistically significant inhibition of growth rate was found at TWA measured concentrations of 1.3 mg/L and higher. Biologically relevant inhibition of growth rate (>10%) was found at TWA measured concentrations of 3.6 mg/L and higher.

The ErC50, ErC10 and NOEC were 6.5, 2.4 and 1.3 mg/L, respectively.

Description of key information

The toxicity to aquatic (freshwater) algae is assessed based on read-across from the close structural analogue Galbascone (CAS# 56973 -85 -4) and the ErC50 and ErC10 values determined at 5.3 and 2.0 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

5.3 mg/L

EC10 or NOEC for freshwater algae:

2 mg/L

Additional information

The acute and long-term algae toxicity of Hexalon is based on information from Galbascone (CAS# 56973 -85 -4). The full read across justification is presented in the Endpoint summary of Aquatic toxicity. In the present section below the executive summary of the algae test with Galbascone is presented.

Galbascone and its algae toxicity

For Galbascone, 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 GLP. Water Accommodated Fractions (WAFs) were separately prepared in closed vessels at loading rates of 0.2, 0.6, 2.0, 6.0, 20 and 60 mg/L applying a 1 -day period of magnetic stirring in closed vessels followed by a 1 -day stabilisation period. Up to 0.6 mg/L the substance was soluble in test medium, higher concentrations formed mixtures, which were siphoned off and used as test solutions. The final test solutions were all referred to as Water Accommodated Fractions (WAFs). Pseudokirchneriella subcapitata was exposed for 72 hours (three replicate flasks per concentration, six replicate flasks per control) under constant illumination and shaking at a temperature of 21 - 23°C. Samples were taken from all treatments at t = 0 , 24 and 72 h and analysed with a validated GC-FID method. At the start of the test, the measured test concentrations were 0.17, 0.52, 1.6, 4.6, 18 and 39 mg/L in the WAFs prepared at loading rates of 0.2, 0.6, 2.0, 6.0, 20 and 60 mg/L, respectively (65 -88% of loading rate). These concentrations remained stable during the first 24 hours of exposure (82-96% of initial) but decreased to 53-75% of initial at the end of the test period. Therefore the TWA measured concentrations were calculated and used for determination of effect concentrations. Loading rates of 0.2, 0.6, 2.0, 6.0, 20 and 60 mg/L corresponded with TWA measured concentrations of 0.14, 0.43, 1.3, 3.6, 15 and 34 mg/L , respectively. Statistically significant inhibition of growth rate was found at TWA measured concentrations of 1.3 mg/L and higher. Biologically relevant inhibition of growth rate (>10%) was found at TWA measured concentrations of 3.6 mg/L and higher. The ErC50, ErC10 and NOEC were 6.5, 2.4 and 1.3 mg/L, respectively.