[2-(cyclohexyloxy)ethyl]benzene

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:

08 July 2016 - 16 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:

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 by EC No. 2016/266 of 7 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

Deviations:

no

Principles of method if other than guideline:

In the final test, the spacing factor was > 3.2. Actual spacing factor was ca. 5.
This was done to be able to derive the NOEC and ECx values for both growth rate and yield.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control at t=0 h, t=24 h, t=48h and t=72 h.
The glass wool used for the preparation of the saturated solution (SS) was retained for possible analysis of the residue.

Volume 2.0 mL
Storage Samples were stored in a freezer until analysis.

At the end of the exposure period, samples were taken from one of the replicates of each 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, 48 and 72 hours of exposure.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: saturated solution
The batch of Phenafleur tested was a clear colourless liquid with a purity of 98.51%. The test item was not completely soluble in test medium at the loading rate initially prepared. No correction was made for the composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying three days of magnetic stirring in closed vessels to reach the maximum solubility of the test item in the test medium. The resulting aqueous mixture was left to stabilize for approximately 1 hour where after the clear and colourless Saturated Solution (SS) was siphoned out through glass wool and used as the highest test concentration. All lower test concentrations were prepared by subsequent dilutions of the SS in test medium.

After preparation, volumes of 120 mL 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.

- Controls: M2-medium without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

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
- Age of inoculum (at test initiation):
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1) 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.

ACCLIMATION
- Acclimation period: 3 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.
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

21-23°C

pH:

start: 7.2-7.5
end: 7.3-7.9

Nominal and measured concentrations:

Based on the results of the combined limit/range-finding test, ErC50 was expected to range between concentrations obtained in 10 and 100% of a saturated solution (SS) prepared at a loading rate of 100 mg/L.
Therefore the following test concentrations were assigned to the definitive test:
Test concentrations: 0.16, 0.80, 4.0, 20 and 100% of an SS prepared at a loading rate of 100 mg/L
Measured test concentrations at test start: 0.0287, 0.152, 0.756, 4.17 and 21.7 mg/L in test solutions 0.16, 0.80, 4.0, 20 and 100% of an SS prepared at a loading rate of 100 mg/L
All concentrations gradually decreased to 66-76% of initial at the end of the test.

EC50 values were expressed in terms of geometric mean measured concentrations (for calculation formula see 'attached background material').

Details on test conditions:

TEST SYSTEM
- Test vessel, fill volume: 120 mL, airtight closed with no headspace to prevent any loss of the test item due to volatilization
- Aeration: no
- Initial cells density: 10^4 cells/mL
- Control end cells density (mean): 115.9 * 10^4 cells/mL
- No. of vessels per concentration (replicates): 3 (plus 2 extra replicates of each test concentration for sampling purposes)
- No. of vessels per control (replicates): 6 (plus 2 extra replicates of the control for sampling purposes)
- Other: 1 to 3 replicates of each test concentration without algae

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)
- Pre-culture and test medium: adjusted M2, i.e. standard M2 according to the OECD 201 Guideline using reverse osmosis purified deionised water (Milli-RO) but with a larger amount of NaHCO3, the addition of HEPES buffer and a lower pH (pH 7.1 +/- 0.3; additional sodium bicarbonate was added to the medium to provide a source of carbon dioxide for algal growth))

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: adjusted M2 (prepared using RO water)
- 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 - continuously in a temperature control vessel

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: light intensity within the range of 67 to 77 µE.m-2.s-1 (TLD lamps)
- Other: 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) :
- 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 cuvettes (path length =10 mm. Algal medium was used as blank.
- Other: Appearance of the cells - At the end of the final test microscopic observations were performed on a replicate containing 20% of the SS and on the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 5
- Range finding study
* Test concentrations: 1.0, 10 and 100% of a saturated solution (SS) prepared at a loading rate of 100 mg/L
- Results used to determine the conditions for the definitive study: yes
The expected EC50 for growth rate inhibition was between concentrations obtained in 10 and 100% of the SS. The expected EC50 for yield inhibition was between concentrations obtained in 1.0 and 10% of the SS.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (July 2016)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 7.1-9.4 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 2.5-4.0 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.65 mg/L

Nominal / measured:

meas. (geom. mean)

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 (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 3.5 mg/L when compared to the control.
- Other:
- Any stimulation of growth found in any treatment: no statistically significant stimulation
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid: yes
- 72h-ErC50: 1.0 mg/L (95% CI: 0.97-1.0 mg/L)
- Other: 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.

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 (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller).

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.

The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Combined limit/range-finding test

Phenafleur, %SS at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.708	0.0319	6
1.0	1.528	0.0304	3	10.6
10	1.432	0.0152	3	16.1
100	0.000	0.0000	6	100.0

 

 

Measured test substance concentrations - final test

 

Analysis of the samples taken at the start of the test showed measured concentrations of 0.029, 0.15, 0.76, 4.2 and 22 mg/L in 0.16, 0.80, 4.0, 20 and 100% of the SS at 100 mg/L, respectively. All concentrations gradually decreased to 66-76% of initial at the end of the test. The measured concentration in the samples taken from 4.0%SS with and without algae were similar. Given this result, the EC₅₀values were expressed in terms of the average exposure concentrations (geometric mean measured concentrations).

 

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

 

Time of sampling^1 [hours]	Percentage of SS^2 [%]	Analysed concentration [mg/L]	Relative to initial [%]
0	0	n.d.
	0.16	0.0287
	0.80	0.152
	4.0	0.756
	4.0^3	0.774
	20	4.17
	100	21.7
24	0	n.d.	n.a.
	0.16	0.0257	90
	0.80	0.139	91
	4.0	0.649	86
	4.0^3	0.697	90
	20	3.69	88
	100	19.6	90
48	0	n.d.	n.a.
	0.16	0.0209	73
	0.80	0.128	84
	4.0	0.639	85
	4.0^3	0.616	80
	20	3.23	77
	100	12.3	57
72	0	n.d.	n.a.
	0.16	0.019^4	66
	0.80	0.112	74
	4.0	0.573	76
	4.0^3	0.591	76
	20	2.89	69
	100	16.5	76

¹      Samples were stored in the freezer (≤ -15°C) until the day of analysis.

²      Percentage of a saturated solution prepared at a loading rate of 100 mg/L.

³      Without algae.

⁴      Estimated value, calculated by extrapolation of the calibration curve.

n.d. Not detected.

n.a.  Not applicable.

 

Table 2: Exposure concentrations

 

Phenafleur, %SS at 100 mg/L	Measured concentration (mg/L)				Average exposure concentration (mg/L)
	t=0h	t=24h	t=48h	t=72h
0.16	0.0287	0.0257	0.0209	0.019	0.023
0.80	0.152	0.139	0.128	0.112	0.13
4.0	0.756	0.649	0.639	0.573	0.65
4.0^1	0.774	0.697	0.616	0.591	0.67
20	4.17	3.69	3.23	2.89	3.5
100	21.7	19.6	12.3	16.5	17

¹Without algae

 

 

Inhibition results – final test

 

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

 

Average conc. Phenafleur (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.580	0.0584	6
0.023	1.581	0.0702	3	-0.1
0.13	1.607	0.0091	3	-1.7
0.65	1.613	0.0241	3	-2.1
3.5	1.390	0.0303	3	12.0*
17	0.247	0.1758	3	84.3*

* Effect was statistically significant

 

Table 4:Percentage inhibition of yield during the final test

 

Average conc. Phenafleur (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	114.9	20.22	6
0.023	115.5	25.64	3	-0.6
0.13	123.0	3.41	3	-7.0
0.65	125.7	9.22	3	-9.4
3.5	63.8	5.76	3	44.4*
17	1.3	0.99	3	98.9*

* Effect was statistically significant

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 did not exceed 35% and CV of average specific growth rates during the whole test period did not exceed 7%

Conclusions:

The 72h-ErC50, ErC10 and NOEC were 8.2, 3.2 and 0.65 mg/L, respectively, based on geometric mean measured concentrations.

Executive summary:

A study was performed to assess the effect of the substance on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201. Preparation of test solutions started with a loading rate of 100 mg/L applying three days of magnetic stirring in closed vessels to reach the maximum solubility of the test item in the test medium, followed by a 1h-stabilisation period whereafter the clear and colourless Saturated Solution (SS) was siphoned out through glass wool and used as the highest test concentration. All lower test concentrations (0.16, 0.80, 4.0 and 20% of the SS) were prepared by subsequent dilutions of the 100% SS in test medium. These concentrations were selected based on the results of the preceding combined limit/range-finding test, performed with 1, 10 and 100 mg/l.  The algae were 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, 48 and 72 h and analysed with a validated HPLC-UV method. At the start of the test, the measured test concentrations were 0.029, 0.15, 0.76, 4.2 and 22 mg/L in the 0.16, 0.80, 4.0, 20 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. These concentrations did not remain stable during the 72 h test period (66-76% of initial at the end of the test period) and therefore geometric mean measured concentrations were calculated and used for determination of effect concentrations. Test solutions at 0.16, 0.80, 4.0, 20 and 100% of the SS prepared at a loading rate of 100 mg/L corresponded with geometric mean measured concentrations of 0.023, 0.13, 0.65, 3.5 and 17 mg/L, respectively. Statistically and biologically (>10%) significant inhibition of growth rate was found at geometric mean measured concentrations of 3.5 mg/L and higher. The ErC50, ErC10 and NOEC were 8.2, 3.2 and 0.65 mg/L, respectively, based on geometric mean measured concentrations.

Description of key information

A study was performed to assess the effect of the substance on the growth of the green algaPseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201.Preparation of test solutions started with a loading rate of 100 mg/L applying three days of magnetic stirring in closed vessels to reach the maximum solubility of the test item in the test medium, followed by a 1h-stabilisation period whereafter the clear and colourless Saturated Solution (SS) was siphoned out through glass wool and used as the highest test concentration. All lower test concentrations (0.16, 0.80, 4.0 and 20% of the SS) were prepared by subsequent dilutions of the 100% SS in test medium. These concentrations were selected based on the results of the preceding combined limit/range-finding test, performed with 1, 10 and 100 mg/l. The algae were 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, 48 and 72 h and analysed with a validated HPLC-UV method. At the start of the test, the measured test concentrations were 0.029, 0.15, 0.76, 4.2 and 22 mg/L in the 0.16, 0.80, 4.0, 20 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. These concentrations did not remain stable during the 72 h test period (66-76% of initial at the end of the test period) and therefore geometric mean measured concentrations were calculated and used for determination of effect concentrations. Test solutions at 0.16, 0.80, 4.0, 20 and 100% of the SS prepared at a loading rate of 100 mg/L corresponded with geometric mean measured concentrations of 0.023, 0.13, 0.65, 3.5 and 17 mg/L, respectively.Statistically and biologically (>10%) significant inhibition of growth rate was found at geometric mean measured concentrations of 3.5 mg/L and higher.The ErC50, ErC10 and NOEC were 8.2, 3.2 and 0.65 mg/L, respectively, based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

8.2 mg/L

EC10 or NOEC for freshwater algae:

3.2 mg/L