2,2'-(cyclohexylimino)bisethanol

Long-term toxicity to fish

Administrative data

Endpoint:

fish short-term toxicity test on embryo and sac-fry stages

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-15 to 2012-10-24

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-(cyclohexylimino)bisethanol

EC Number:

224-809-5

EC Name:

2,2'-(cyclohexylimino)bisethanol

Cas Number:

4500-29-2

Molecular formula:

C10H21NO2

IUPAC Name:

2,2'-(cyclohexylimino)bisethanol

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: A limit concentration of 116.27 mg test item/L, corresponding to 100 mg active substance/L, was tested.
- Sampling method: Samples of test media including control were taken from alternating test replicates in 3 sampling intervals from freshly prepared and corresponding 24 h old test solutions.
- Sample storage conditions before analysis: All samples were stored at room temperature before preparation and before analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Directly weighing of the stock solution and agitation.
- Eluate: Dilution water
- Differential loading: None, limit test concentration of 116.27 mg test item/L
- Controls: 40 eggs in dilution water (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Gnathostoma, Pisces, Osteichthyes, Teleostei, Clupeiformes, Cyprinidae
- Source: All fish eggs used in the test were gained at DR.U.NOACK-LABORATORIEN from a single brood stock (supplier: Umweltbundesamt, Schichauweg 58, 12307 Berlin, Germany).

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Adult zebrafish were kept in separate aquaria. About 15 minutes before start of artificial dawning (1 h) rectangular dishes (26 cm x 14 cm x 6 cm), covered with a stainless steel mesh and provided with artificial plants, were introduced into the aquaria. After approx. 3 h the glass dishes were gently removed. About 150 eggs were taken and immediately distributed to the test solution and dilution water (for control).
- Subsequent handling of eggs: After approximately 4 h eggs were checked for fertilization. Under a stereo microscope every embryo was checked for its blastomer phase. Eggs with only a 2 cell blastomer were regarded not to be fertilised. These eggs as well as coagulated eggs were discarded. Only fertilised eggs with more than 2 cells were introduced in the test vessels. 10 eggs were introduced per replicate.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

9 d

Test conditions

Hardness:

Total hardness 45 mg CaO3/L (control), 53 mg CaCO3 (limit test concentration)

Test temperature:

Period of measurements 2012-10-15 to 2012-10-23
Minimum temperature [°C] 24.4
Maximum temperature [°C] 25.9
Mean temperature
± Standard deviation [°C] 25.6 ± 0.2

pH:

pH Values in the Test Media

Study day Test media pH
Nominal concentration
test item / active substance
[mg/L]
Control 116.27 / 100
0 new 7.28 9.44
1 old 7.41 8.24
new 7.41 9.13
2 old 7.43 8.26
new 7.46 9.15
3 old 7.32 8.02
new 7.44 8.97
4 old 7.39 8.15
new 7.46 9.12
5 old 7.75 8.12
new 7.89 9.11
6 old 7.61 8.28
new 7.66 8.97
7 old 7.77 8.43
new 7.15 9.06
8 old 7.59 8.29
Mean 7.50 8.67
SD ± 0.20 0.48
Min. 7.15 8.02
Max. 7.89 9.44

Dissolved oxygen:

Dissolved Oxygen in Percent Air Saturation Value

Study day Test media Dissolved Oxygen [%]
Nominal concentration
test item / active substance
[mg/L]
Control 116.27 / 100
0 new 100 100
1 old 100 100
new 100 100
2 old 100 98
new 100 100
3 old 100 98
new 100 100
4 old 99 98
new 100 99
5 old 98 96
new 99 98
6 old 98 97
new 100 100
7 old 98 97
new 100 100
8 old 97 98
Mean 99 99
SD ± 1.01 1.35
Min. 97 96
Max. 100 100

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Crystallisation dishes (inner diameter 13.5 cm, water height about
5 cm) were used. The volume of the test media in the dishes was about 500 mL.
- Aeration: No aeration was provided. Semi-static conditions with renewal of the test media in 48 h intervals was performed.
- No. of fertilized eggs/embryos per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Tap water of local origin was used. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5


OTHER TEST CONDITIONS
- pH: 6 – 8
- Photoperiod: 16 h photoperiod daily
- Light intensity: 0.1 - 10 µmol photons • m-2 • s-1on water surface (diffuse light)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Biological parameters Observations were made daily.

Hatched eggs The number of hatched eggs was determined daily until 5 days post-hatch.

Post hatch period Per definition the post hatch period begun when at least 80 % of all fertilized and living embryos in the control group have hatched (day 3 of the study).

Mortality Criteria for mortality vary according to life stage:

For eggs: Mortality as discerned by a distinct change in coloration or a marked loss of translucency and change in coloration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance, was checked daily. Dead eggs were discarded.

For embryos: Absence of body movement and/or heart-beat, change in coloration. Dead embryos were discarded.

For larvae: Immobility and/or absence of respiratory movement and/or absence of heart-beat (as far as visible) and/or lack of reaction to mechanical stimulus.

Further effects
Abnormal appearance and behaviour were also recorded.The number of larvae or fish showing abnormality of body form and/or pigmentation and the stage of yolk-sac absorption was recorded. Abnormal animals were only removed from the test vessels on death. Abnormalities, e.g. hyperventilation, uncoordinated swimming, swim-up behaviour, atypical quiescence, and atypical feeding behaviour will also be recorded by visually inspecting each replicate.

Measurement of fish size At the end of exposure (post-hatch day 5) the fish were euthanized in a Benzocaine solution and the total length of all survivors was measured to the nearest 0.001 mm with a microscope camera and corresponding software (ImageFocus, EUROMEX BV).

Measurement of
dry body weight The mean fish dry weight from pooled survivors per replicate wasdetermined at the end of exposure (post-hatch day 5, study day 8). The fish were dried for 24 h at 60 °C. Dry biomass weight was measured to the nearest 0.001 mg.

Chemical parameters

Water quality and Temperature, pH value and oxygen saturation were measured
light intensity daily in freshly prepared and old test media in one replicate of the
measurements limit concentration and the control. Temperature was recorded continuously in the dilution water by a datalogger.
Total hardness was measured at the beginning of exposure from one replicate of the limit concentration and the control, respectively. Chlorine and TOC were measured at the beginning of the test from the dilution water. The light intensity on the surface of the test vessels was measured at test start.



VEHICLE CONTROL PERFORMED: no


Overall Survival in the Preliminary Test (24 – 72 h)
(15 eggs / concentration)
Nominal test item concentration
[mg/L] Effect n/15 Overalll Survival 72 h[%]
24 h 48 h 72 h
200
(pH control) Survival (overall) 14 14 14 93
200 Survival (overall) 15 15 15 100
20.0 Survival (overall) 15 15 15 100
2.00 Survival (overall) 13 13 13 87
Control Survival (overall) 14 14 14 93

Results and discussion

Effect concentrations

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Hatch, growth, length, fry weight, post hatch survival, overall survial

Remarks on result:

other: Limit test

Details on results:

- Mortality/survival at embryo and larval stages:
The post hatch success in all control replicates met the guideline criteria. The fry survival (post hatch success) at the end of the study was 98 % in the control group and 100 % in the limit test concentration, respectively.
One way analysis of variance and Dunnett’s test were carried out for the results of post hatch day 5 (end of the study). No statistically significant difference was found for the limit test concentration.
- Overall mortality/survival:
Overall survival at the end of the study was 95 % in the control group and 93 % in the limit test concentration, respectively . One way analysis of variance and Dunnett’s test were carried out for the results of overall survival on post hatch day 5 (end of the study). No statistically significant difference was found for the limit test concentration.
- Days to hatch and numbers hatched:
Egg hatch began on study day 3 in the control and the limit concentration and continued until study day 4. Study day 3 was determined to be post hatch day 0 (PHD 0) with a control hatching rate of 95 %.
Statistical procedures (one way analysis of variance) were applied for study days 3 and 4 (post hatch days 0 and 1). A statistically significant difference was found on post hatch day 0 for the limit concentration. However, this effect was transient and considered to be biologically not significant.
- Data for length and weight of surviving fish:
The fry growth until study day 8 (post hatch day 5), expressed as length and dry weight, was determined.
One way analysis of variance and Dunnett’s test were carried out for the results of post hatch day 5 (end of the study). For the length and weight data no statistically significant difference was found for the limit test concentration.
- Type of and number with morphological abnormalities:
On study day 6 and 7 fish larvae in the limit concentration showed abnormal swimming behaviour. Study day 6: 7 of 37 larvae in the limit concentration showed quiescence marked by abnormally low activity or inactivity, as well as abnormally long remaining on the bottom of the test vessel.
Study day 7: 14 of 37 larvae in the limit concentration showed same abnormal behavior as observed on study day 6. Since this effect was transient it is considered to be biologically not significant.

Reported statistics and error estimates:

The concentrations leading to 0 and 100 % mortality (LC0 and LC100) were determined directly from the test results. LC50-values have not to be calculated in a limit test.
One way analysis of variance (ANOVA) was used for NOEC/LOEC calculations. When running a one way analysis of variance a normality test and an equal variance test were done first. For the parameters hatch, growth (length and dry weight), post hatch survival and overall fry survival (mortality), the following statistical tests were conducted:
Hatching data of study day 3 (post hatch day 0) and study day 4 (post hatch day 1) were analysed with ANOVA.
Dry weight data, length data, post hatch survival and overall survival (mortality) data of study day 8 (post hatch day 5) were analysed with ANOVA. Normality test failed for post hatch survival. Equal variance test failed for length data. No transformations were carried out with these data because the data sets were not estimated to follow a normal distribution.
These statistical analyses were conducted with conclusions of statistical significance based on a 95 % confidence level. The a-value (acceptable probability of incorrectly concluding that there is a difference) was 0.05.

Any other information on results incl. tables

Egg Hatch / Hatching Time

Nominal concentration [mg/L]		Replicate	Egg hatch [%]
Test item	Active substance		Post hatch day -2 (Study day 1)	Post hatch day -1 (Study day 2)	Post hatch day 0 (Study day 3)	Post hatch day 1   (Study day 4)	Post hatch day 2   (Study day 5)
116.27	100	1	0	0	60	80	80
		2	0	0	90	100	100
		3	0	0	70	100	100
		4	0	0	80	90	90
		Mean	0	0	75   (+)	93	93
Control		1	0	0	100	100	100
		2	0	0	90	100	100
		3	0	0	90	90	90
		4	0	0	100	100	100
		Mean	0	0	95	98	98

Overall Survival and Mortality on Study Day 8 (Post Hatch Day 5)

Nominal concentration [mg/L]		Replicate	Live fry on post hatch day 5 (Study day 8) n/10	Overall survival [%]	Mortality [%]
Test item	Active substance
116.27	100	1	8	80	20
		2	10	100	0
		3	10	100	0
		4	9	90	10
		Mean	9	93	8
Control		1	9	90	10
		2	10	100	0
		3	9	90	10
		4	10	100	0
		Mean	10	95	5

Fry Growth: Length and Dry Weight on Study Day 8 (Post Hatch Day 5)

Nominal concentration [mg/L]		Replicate	Number of fish larvae	Post hatch day 5 (Study termination)
Test item	Active substance			Mean length per fish larvae [mm]	Pooled  dry weight [mg]	Mean dry weight per fish larvae [mg]
116.27	100	1	9	4.037	0.374	0.0468
		2	10	4.017	0.480	0.0480
		3	9	4.057	0.478	0.0478
		4	10	4.045	0.398	0.0442
		Mean		4.039	0.433	0.0467
Control		1	8	4.082	0.344	0.0382
		2	10	3.994	0.516	0.0516
		3	10	4.060	0.422	0.0469
		4	9	3.973	0.472	0.0472
		Mean		4.027	0.439	0.0460

Percent Swim-up of Hatched Fry

Nominal concentration [mg/L]		Replicate	Swim-up [%]
Test item	Active substance		Post hatch day 0 (Study day 3)	Post hatch day 1 (Study day 4)	Post hatch day 2 (Study day 5)	Post hatch day 3 (Study day 6)	Post hatch day 4   (Study day 7)
116.27	100	1	0	0	88	75	50
		2	0	0	70	70	70
		3	0	0	90	90	70
		4	0	0	89	89	56
		Mean	0	0	84	81	62
Control		1	0	10	90	100	100
		2	0	0	70	100	100
		3	0	22	89	100	100
		4	0	0	90	100	100
		Mean	0	8	85	100	100

Maximum Hatch of Larvae, Vital Larvae and Post Hatch Survival on Study Day 8
(Post Hatch Day 5)

Nominal concentration [mg/L]		Study day with maximum hatch	Replicate	Hatched larvae on study day with maximum hatch n/10	Vital larvae on study day 8 (PHD 5) n/10	Post hatch survival on study day 8 (PHD 5) [%]
Test item	Active substance
116.27	100	4                   (PHD 1)	1	8	8	100
			2	10	10	100
			3	10	10	100
			4	9	9	100
			Mean	9	9	100
Control		4                   (PHD 1)	1	10	9	90
			2	10	10	100
			3	9	9	100
			4	10	10	100
			Mean	10	10	98

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

2,2’-(Cyclohexylimino)diethanol did not cause significant effects on the embryo and sac-fry stages of zebrafish when tested with the limit concentration of 116.27 mg test item/L, corresponding to 100 mg active substance/L. Based on the parameters hatch, growth (expressed as length and dry weight), post hatch and overall survival (mortality) the overall NOEC (post hatch day 0 – 5) was 116.27 mg test item/L corresponding to 100 mg active substance/L. The overall LOEC was > 116.27 mg test item/L, corresponding to > 100 mg active substance/L.

Executive summary:

The effects of the test item 2,2’-(Cyclohexylimino)diethanol(batch: ESD0011452) to the embryo and sac-fry stages of fish (Zebrafish / Danio rerio) were determined according to OECD Guideline 212 from 2012-10-15 to 2012-10-24 at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany.

A semi-static test procedure with renewal of the test media in 24 h intervals was performed with the nominal limit concentration of of 116.27 mg test item/L, corresponding to 100 mg active substance/L.

The test was started by placing fertilized eggs in the test vessels and lasted 8 days (5 days post-hatch). 40 eggs of Danio rerio were exposed to the limit concentration and the control (4 replicates with 10 eggs each), respectively.

On day four 95 % of the control larvae had hatched. Therefore, study day 3 had been defined as post hatch day 0 (= PHD 0).

Different toxic endpoints were determined: egg hatch, time to hatch, fry growth (expressed as length and weight), morphological and behavioural effects, post hatch survival, overall fry survival and mortality, respectively. The results of the named parameters were checked for statistically significant differences. The NOEC, LOEC and LC-values were determined based on the statistical results.

The concentration of the test item and control were analytically verified via LC-MS/MS in 3 sampling intervals: from freshly prepared media on days 0, 3 and 7 and from corresponding 24 hours old media on days 1, 4 and 8. The measured concentrations of 2,2’-(Cyclohexylimino)diethanolin freshly prepared media were in the range of 101 - 105 % of the nominal values and 104 – 114 % in the corresponding 24 h aged test media.

All effect levels are given based on the nominal concentrations of the test item 2,2’-(Cyclohexylimino)diethanol

Hatch, Growth, Fry survival: NOEC, LOEC

                      based on nominal concentrations [mg/L]

	Test item		Active substance
Parameter	NOEC	LOEC	NOEC	LOEC
Hatch	116.27	> 116.27	100	> 100
Length	116.27	> 116.27	100	> 100
Weight	116.27	> 116.27	100	> 100
Post hatch survival	116.27	> 116.27	100	> 100
Overall survival	116.27	> 116.27	100	> 100
Overall NOEC and LOEC	116.27	> 116.27	100	> 100

Table 2 :      NOEC and LC-Values with 95 % Confidence Interval on Study Day 8 (Post Hatch Day 5)

                      based on nominal concentrations [mg/L]

	Test item	Active substance
LC50	> 116.27	> 100
LC100	> 116.27	> 100
Lowest test item concentration with 100 % mortality on Study day 8
LC0	116.27	100
Highest test item concentration with 0 % mortality on Study day 8