Reaction product of o-cresol and camphene, obtained by addition, hydrogenation and distillation

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:

29 Jun - 10 Sep 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:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office of Public Health, Consumer protection directorate, Notification authority for chemicals, Bern, Switzerland

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The control and loading rates of 15, 30, 45, 67 and 100 mg/L were analysed analytically.
- Sampling method: Duplicate samples without algae were taken from each loading rate and the control at the start of the test. At the end of the test (after 72 h), samples (containing algae) were taken in duplicate from all loading rates and from the control. For sampling from the aged test media, the contents of the respective replicates were combined prior to sampling.
- Sample storage conditions before analysis: All samples were stored frozen (at -20 ± 5 °C) immediately after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Accommodated Fractions (WAFs) were prepared individually. The test item was mixed into test water by intensive stirring for 96 h in closed vessels in the dark to dissolve the different components of the test item as far as possible within a reasonable time period. After stirring, the emulsions were filtered through a 0.45 μm membrane filter (Whatman, NC45). As a pre-caution, the filter was pre-conditioned with 200 mL filtrates to avoid losses of dissolved test item due to adsorption on the filter material. The undiluted filtrates were tested as WAFs.
- Eluate: no
- Differential loading: yes
- Controls: yes, test medium control

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Germany).
- Method of cultivation: An inoculum culture was set up four days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 °C

pH:

7.4 - 8.0

Nominal and measured concentrations:

nominal: control, 15, 30, 45, 67 and 100 mg/L
measured: < LOQ, 0.36, 0.69, 1.1, 1.2 and 1.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: covered with a glass lid
- Material, size, headspace, fill volume: glass, 75 mL, headspace 45 mL, fill volume: 30 mL
- Aeration: constantly shaken
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: LED light, approx. 72 μE s-1 m-2

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: electronic particle counter (Cell Counter CASY TT, OLS, Bremen, Germany) for cell number in pre-culture. The algal biomass in the samples was determined by fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd, Wokingham Berkshire/UK). The measurements were performed at least in duplicate at an excitation of 440 nm and emission of 680 nm.
- Cell shape: Cell shape and size was determined by visual inspection.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5
- Range finding study:
- Test concentrations: control, 1.0, 10. 100 mg/L loading rate
- Results used to determine the conditions for the definitive study: No inhibition of growth was recorded at any of the loading rates tested. Thus, a limit test with a loading rate of 100 mg/L was performed first. Since significant inhibition of growth was recorded in the first main test a second main test with loading rates of 15, 30, 45, 67 and 100 mg/L was tested.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 14-22 mg/L

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the loading rate of 100 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this test concentration.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Test solutions were clear and colorless.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 1.0 mg/L (tested May 2017)
- Other: Sensitivity of the test system was within the range recommended by the guideline (EC50 (72 h) for the growth rate 0.9-1.5 mg/L).

Reported statistics and error estimates:

The EL10, EL20 and EL50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated as far as possible by Probit Analysis using linear maximum likelihood regression. For the determination of the LOEL and NOEL, the average growth rate and yield at the test concentrations were compared to the control values by the Williams t-test. Statistical analysis was performed using ToxRat Professional®.

Table 1: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	221	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	15%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	0.8%	yes

Table 2: Inhibition of growth

Loading rate [mg/L]	Average growth rate µ (day-1) and inhibition µ (Ir)
	0-24 h		0-48 h		0-72 h
	µ	Ir [%]	µ	Ir [%]	µ	Ir [%]
Control	1.508	0.0	1.778	0.0	1.799	0.0
15	1.351*	10.4	1.721*	3.2	1.801	-0.1
30	1.237*	18.0	1.658*	6.8	1.739*	3.3
45	1.089*	27.8	1.555*	12.6	1.676*	6.8
67	1.277*	15.3	1.599*	10.1	1.666*	7.4
100	0.830*	45.0	1.519*	14.6	1.652*	8.2

*: Mean value statistically significantly lower than in the control (according to Williams t-test one-sided smaller,α= 0.05).

Table 3: Inhibition of Yield

Loading rate [mg/L]	Yield Y (E+04) and inhibition of Y (Iy)
	0-24 h		0-48 h		0-72 h
	Y	Iy [%]	Y	Iy [%]	Y	Iy [%]
Control	2.18	0.0	21.05	0.0	135.63	0.0
15	1.77*	19.0	18.67*	11.3	136.61	-0.7
30	1.53*	30.0	16.40*	22.1	113.13*	16.6
45	1.23*	43.9	13.23*	37.2	93.67*	30.9
67	1.61*	26.4	14.55*	30.9	90.92*	33.0
100	0.80*	63.3	12.27*	41.7	87.04*	35.8

*: Mean value statistically significantly lower than in the control (according to Williams t-test one-sided smaller,α= 0.05).

Table 4: Measured concentrations of WAF loading rates

Sampling Day/ Sample Age	Water Accommodated Fractions, Loading Rate of Test Item	Measured Concentration  of Test Item x	Sample Preparation Factor F	Determined Concentration of Test Item  c	% of Initially Measured
[day/hours]	[mg/L]	[mg/L]		[mg/L]	[%]
0/0	Control	n.d.	0.1	<LOQ	-
(fresh)	15	3.64	0.1	0.364	-
	30	6.93	0.1	0.693	-
	45	11.0	0.1	1.10	-
	67	11.6	0.1	1.16	-
	100	10.3	0.1	1.03	-
3/72	Control	n.d.	0.1	<LOQ	-
(aged)	15	0.654	0.1	0.0654*	18
	30	2.11	0.1	0.211	30
	45	2.83	0.1	0.283	26
	67	2.39	0.1	0.239	21
	100	4.07	0.1	0.407	39

n.d. = no test item detected

n.a. = not applicable

* Estimated value, the determined concentration was below the LOQ.

LOQ: 0.115 mg/L

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Conclusions:

The 72-hour EL10 and EL50 values for the inhibition of growth rate were found to be >100 mg/L. All effect values were based on the nominal loading rate.

Description of key information

ErL10 (72 h) >100 mg/L (nominal, OECD 201)
ErL50 (72 h) >100 mg/L (nominal, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

One experimental study is available investigating the toxicity of the test item to aquatic algae. The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour test according to OECD 201 (2011) under GLP conditions. Since the test item is a complex mixture (UVCB) consists of several constituents with differently low solubilities, Water Accommodated Fractions (WAFs) with the loading rates of 15, 30, 45, 67 and 100 mg/L were tested to assess the toxicity of the test item to algae. Additionally, a control group (test water without test item) was tested in parallel. The actual test concentrations were verified by GC/FID measurement at the start and end of the test. At the start of the test, the measured concentrations of the analyzed components of the test item in the test media were 0.36, 0.69, 1.1, 1.2 and 1.0 mg/L, respectively. At the three highest loading rates (45, 67 and 100 mg/L), the analyzed test item concentrations were in the same range of 1.0 to 1.2 mg/L, showing a plateau and demonstrating that the maximum concentration (sum of peaks) of dissolved test item was reached under the conditions of the test. The measured concentrations at the end of the test were in the range of 18 and 39 % of the initially measured values. Inhibition of growth rate was only 8.2 % at the highest loading rate of 100 mg/L resulting in an 72-hour ErL10/50 of >100 mg/L. Since WAFs were tested all reported biological results were based on the loading rates of the test item. The preparation of the WAFs and procedure of evaluation of data of WAFs is based on recommendations of the OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.