2,2'-(cyclohexylimino)bisethanol

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-08-03 to 2011-08-05

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

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

Details on test material:

- Name of test material (as cited in study report): 2,2'-(Cyclohexylimino)diethanol
- Physical state: Clear liquid
- Analytical purity: 86 %
- Purity test date: 2011-04-14
- Lot/batch No.: ESD0011452
- Expiration date of the lot/batch: 2013-02-28
- Stability under test conditions: Stable in water and light at room temperature
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The active ingredient 2,2’-(Cyclohexylimino)diethanol were analytically verified by LC-MS/MS in the fresh media (0 h) and old media (48 h) of all concentration levels, the pH-control and the control.
- Sampling method: For the old media, separate replicates without test organisms were prepared and incubated under test conditions. The method was validated according to SANCO 3029/99 rev.4 (2000-07-11).
- Sample storage conditions before analysis: All samples were stored at room temperature.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 400 mg/L was freshly prepared with dilution water
- Eluate: Dilution water
- Differential loading: 25.0 - 50.0 - 100 - 200 - 400 mg/L, prepared with dilution
water.
- Controls: The highest test concentration was adjusted to a pH in the range 7.0  0.2 by addition of 1 M HCl and tested under the same conditions as the test and control groups.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20+/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µE*m-2*s-1. Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L. Culture Feeding: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata, Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 106 cells/mL.
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water: 247 (mg CaCO3/L)

Test temperature:

21.3 °C.

pH:

Water Quality Parameters at Test Start (0 h, Fresh Media)
2,2’-(Cyclohexylimino)diethanol
Nominal
Test Item Concentration
[mg/L] pH-Value



400
(pH-control)* 6.98
400 9.77
200 9.50
100 9.28
50.0 8.98
25.0 8.70
Control 7.94

Water Quality Parameters at Test End (48 h, Old Media)
2,2’-(Cyclohexylimino)diethanol

Nominal
Test Item Concentration
[mg/L] pH-Values

Replicates
1 2 3 4
400
(pH-control)* 7.56 7.57 7.53 7.53
400 8.48 8.43 8.62 8.85
200 8.17 8.16 8.25 8.42
100 7.87 7.88 7.87 7.83
50.0 7.79 7.81 7.81 7.82
25.0 7.68 7.76 7.76 7.72
Control 7.77 7.79 7.67 7.66

Dissolved oxygen:

2,2’-(Cyclohexylimino)diethanol
Nominal
Test Item Concentration
[mg/L] Dissolved
O2-Concentration

[mg/L]
400
(pH-control)* 8.42
400 8.37
200 8.42
100 8.41
50.0 8.44
25.0 8.47
Control 8.48

2,2’-(Cyclohexylimino)diethanol

Nominal
Test Item Concentration
[mg/L] Dissolved O2 -Concentration
[mg/L]
Replicates
1 2 3 4
400
(pH-control)* 8.03 8.32 7.78 6.89
400 8.26 8.33 8.31 8.32
200 8.23 7.95 8.27 8.45
100 8.47 8.22 8.29 8.00
50.0 8.47 8.53 8.53 8.56
25.0 8.50 8.67 8.52 8.29
Control 8.43 8.46 8.37 8.41

Salinity:

Freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): One application at test start, static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to Council Regulation (EC) No. 440/2008 Method C.2, Appendix 2
Component Concentration [mg/L]
KCl 5.76
NaHCO3 64.8
CaCl2 * 2 H2O 294
MgSO4 * 7 H22O 123
pH-value: 7.8 +/- 0.2
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: At test start and test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
IImmobility was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim within 15 sec after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Range finding study
Immobilization Rates of the Preliminary Test after 24 and 48 h of Exposure
(n = 20, divided into 2 replicates with 10 daphnids each)
2,2’-(Cyclohexylimino)diethanol
IMMOBILIZATION [%]
Nominal
Test Item Concentration
[mg/L] 24 h 48 h
Replicates Replicates
1. 2. MV 1. 2. MV
100* 0 0 0 50 70 60
10 0 0 0 0 0 0
1 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

140 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: (121-165)

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

52.9 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: (38-71 mg/L)

Details on results:

- Behavioural abnormalities: None
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50 : 1.77 mg/L (CI 1.63 - 1.96 mg/L)

Reported statistics and error estimates:

EC10- and EC50-values were calculated by sigmoidal dose-response regression. The confidence intervals were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism. The concentration effect relationships are shown graphically.

Any other information on results incl. tables

Immobilization Rates of the Definitive Test after 24 and 48 h of Exposure

                 (n = 20, divided into 4 replicates with 5 daphnids each)

2,2’-(Cyclohexylimino)diethanol	IMMOBILIZATION [%]
Nominal Test Item Concentration [mg/L]	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
400 (pH-control)*	20	20	0	20	15	60	40	80	80	65
400	60	80	80	80	75	100	100	100	100	100
200	0	0	0	0	0	40	60	60	80	60
100	0	0	0	0	0	40	20	40	20	30
50.0	0	0	0	0	0	0	0	0	0	0
25.0	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item 2,2’-(Cyclohexylimino)diethanol the
48-h EC50 for Daphnia magna was estimated to be 163 mg/L (95 % confidence limits: 141 - 192 mg/L).
The NOEC after 48 h was determined to be 50.0 mg/L as the highest tested concentration level with no biologically significant effect. The LOEC after 48 h was determined to be 100 mg/L.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS) the effects of the test item 2,2’-(Cyclohexylimino)diethanol(batch number: ESD0011452) were determined according to OECD 202 (2004) from 2011-08-03 to 2011-08-05 at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany

The study was conducted under static conditions over a period of 48 h with 5concentrations of the test item 2,2’-(Cyclohexylimino)diethanolranging from 25.0 to 400 mg/L set up in a geometric series with a dilution factor of 2. Twenty daphnids were exposed to each concentration level, the pH-control and the control.

The active ingredient 2,2’-(Cyclohexylimino)diethanolwas analytically verified by LC-MS/MS in the fresh (0 h) and old media (48 h) of all concentration levels, the pH-control and the control.
The recoveries of the active ingredient 2,2’-(Cyclohexylimino)diethanolwere in the range of 107 to 114 % of the nominal values in the fresh media (0 h) and 101 to 115 % in the old media (0 h)demonstrating that concentrations of 2,2’-(Cyclohexylimino)diethanolwere successfully maintained for the duration of the test. The analytical results are presented in part 4.2.2.

All effect values, presented in Table 1, are based on the nominal concentrations of the test item.

 

The water quality parameters (pH-value and dissolved oxygen concentration), measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

EC₁₀-, EC₅₀- (with Confidence Interval), EC₁₀₀ - Values, NOEC and LOEC

              (based on the nominal concentrations of the test item 2,2’-(Cyclohexylimino)diethanol)

	Test Duration [h]	2,2’-(Cyclohexylimino)diethanol Nominal Concentration of the Test Item [mg/L]	Confidence Interval p = 95% [mg/L]
EC10	24	304	296 - 312
	48	61.5	44.0 - 82.4
EC50	24	365	358 - 372
	48	163	141 - 192
EC100	24	> 400	---
	48	400	---
NOEC	48	50.0	---
LOEC	48	100	---