Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butan-2-one and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

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:

17 Nov 2017 - 19 Dec 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)

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

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

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: WAFs individually prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L
- Sampling method: Samples (4.0 mL) for possible analysis were taken from all test concentrations and the control at t=0 h, t=24 h and t=72 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates with algae 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:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with loading rates individually prepared ranging between 1.0 and 100 mg/L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle for approximately one hour. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were siphoned off and used as test solutions. All test solutions were clear and colorless 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 1 x 10^4 cells/mL.
- Controls: Test medium without test item or other additives.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- 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.
- Pre-culture: 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.
- Stock culture medium: M1, according to NPR 6505
- Pre-culture medium and test medium: M2, according to OECD201.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22 - 23°C

pH:

At t=0 h: 8.0-8.3
At t=72 h: 8.2-8.5

Nominal and measured concentrations:

Nominal: WAFs individually prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L
Initial measured concentrations: 0.93, 3.1, 8.5, 28 and 76 mg/L. The measured concentrations decreased to 27 – 32% of initial at the end of the test. Based on these results, the TWA concentrations were calculated to determine the effect parameters. See Table 4 in 'Any other information on results' for measured concentrations throughout the test and the calculated TWA concentration.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, capped, containing 50 mL of test solution
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 257 x 10^4 cells/mL
- 3 replicates of each test concentration,
- 6 replicates of the control,
- 1 extra replicate of each test group for sampling purposes after 24 hours of exposure,
- 1 or 2 replicates of each test concentration without algae.
- Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, formulated using tap-water purified by reverse osmosis.
- Culture medium different from test medium: yes, M1
- 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
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod, light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 83 to 86 µE.m-2.s-1

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: 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 (path length = 10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Other: At the end of the final test microscopic observations were performed on the WAF prepared with a loading rate of 10 mg/L to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Combined Limit/Range-Finding test concentrations: WAFs individually prepared at loading rates of 1.0, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Jan 2018)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval 20-24 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Based on statistical significance

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Based on biological relevance

Details on results:

Growth rates were in the range of the controls at the two lowest test concentrations during the 72-hour test period, whereas the growth rates of algae exposed to concentrations of 4.3 mg/L and higher were increasingly reduced. Statistically significant inhibition of growth rate was found at test concentrations of 4.3 mg/L and higher. However, the effects observed at 4.3 mg/L were considered not biologically relevant and therefore, the NOEC based on biological relevance was set to 4.3 mg/L.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a TWA concentration of 4.3 mg/L when compared to the control.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50 for growth rate inhibition (72h-ERC50) was 0.86 mg/L with a 95% confidence interval ranging from 0.84 to 0.88 mg/L.
- The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Calculation of significant effects: Step-down Jonckheere-Terpstra Test Procedure, 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 versus the logarithms of the corresponding TWA concentrations of the test item. The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 4: Measured Concentrations Versus Nominal Concentrations

POLYAMBROL Nominal conc. (mg/L)	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72 h
1.0	0.925	0.520	0.259	0.48
3.2	3.10	2.06	0.827	1.7
10	8.48	4.48	2.46	4.3
10^WA	8.53	3.38	2.50	3.7
32	28.3	13.8	8.01	14
100	75.6	41.7	23.8	40

^WA– without algae.

Table 5: Growth Rate And Percentage Inhibition For The Total Test Period

POLYAMBROL TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.849	0.0191	6
0.48	1.854	0.0147	3	-0.3
1.7	1.838	0.0253	3	0.6
4.3	1.682	0.0234	3	9.0^#
14	1.390	0.0093	3	24.8*
40	0.347	0.2337	3	81.2*

* - effect was statistically significant,

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

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, POLYAMBROL reduced growth rate of this fresh water algae species significantly at TWA concentrations of 1.7 mg/L and higher. The EC50 for growth rate inhibition (72h-ERC50) was 22 mg/L with a 95% confidence interval ranging from 20 to 24 mg/L. The 72h-NOEC for growth rate inhibition was 4.3 mg/L based on biological relevance and 1.7 mg/L based on statistical significance.

 

Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to Water Accommodated Fractions of the test substance individually prepared at loading rates of 1.0, 3.2, 10, 3.2 and 100 mg/L. The initially measured concentrations were 0.93, 3.1, 8.5, 28 and 76 mg/L. The measured concentration decreased to 27 -32% of initial at the end of the test. Therefore, the Time Weighted Average concentrations were calculated to determine the effect parameters. TWA concentrations were 0.48, 1.7, 4.3, 14 and 40 mg/L. A dose-related increas of growth rate inhibition was observed at the end of the test. The EC50 for growth rate inhibition was 22 mg/L (95% confidence interval: 20 -24 mg/L). The 72 -h NOEC was 4.3 mg/L and 1.7 mg/L, based on biological relevance and statistical significance, respectively. The study met all validity criteria, and is considered reliable without restriction.

Key value for chemical safety assessment

EC50 for freshwater algae:

22 mg/L

EC10 or NOEC for freshwater algae:

4.3 mg/L