1,6-Hexandiammonium dihydroxide, N,N'-bis-(2-hydroxypropyl)-N,N,N',N'-tetramethyl

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 22 JUL 2002 to 26 JUL 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

pH-value in water 13-14

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg a.i./L (238 mg/L test item) and control
- Sampling: from freshly prepared media after 0, 24, 48 and 72 h and from old media after 24, 48, 72 and 96 h
- Sample storage conditions before analysis: not reported

Vehicle:

no

Details on test solutions:

TEST SOLUTION
- Test concentration: 238 mg/L corresponding to 100 mg a.i./L
- Preparation of stock solution: direct weighing
- Dispersion treatment: test medium was mixed with an ultraturrax (1 min, 20500 rpm)

- Equipement:
Laboratory blender "T 25", IKA -LABORTECHNIK

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Source: Origin AQUARIUM AM AEGI, Hannover, Germany. All fish used in the test originated from the same delivery of the supplier.
- Age at study initiation (mean and range, SD): not reported
- Length at study initiation (length definition, mean, range and SD): 2.96 cm
- Weight at study initiation (mean and range, SD): 0.28 g

ACCLIMATION
- Acclimation period: at least 12 days of acclimatisation
- Acclimation conditions (same as test or not): Same as test conditions: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine; pH-value: 6.0 - 8.5; Hardness 40 - 180 mg CaCO3 / L
- Holding: Holding was performed at the test facility at 23 ± 2 °C and diffuse light (0.1-10 µmol/m^2*s, natural photoperiod). Water was changed at least once per week.
The dissolved oxygen concentration was more than 80% of the air saturation value.
- Type and amount of food during acclimation: Trouvit 40/2; MILKIVIT, Bürgheim / Food was provided 3 times per week. The amount of food was 4% of the fish body weight per feeding day. The test fish were not fed 24h before test start.
- Feeding frequency during acclimation: yes
- Health during acclimation (any mortality observed): mortality < 5 % within the last 7 days before the study started
- Medication: No disease treatments were administered throughout holding and testing.

FEEDING DURING TEST
- no feeding

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

58-67 mg/L as CaCO3

Test temperature:

21.4-22.1 °C

pH:

7.18-10.07

Dissolved oxygen:

95-100% of the air saturation value

Nominal and measured concentrations:

Nominal test concentration: 100 mg a.i./L (238 mg/L test item)
Measured test concentration: see any other information on results.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass-aquaria loosely covered by glass tops were used.
- Test volume: The test volume was 10 L per vessel.
- Renewal rate of test solution (frequency/flow rate): after 24 hours (daily)
- Aeration: Gentle aeration was provided.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Speciman: Fish were introduced randomly to individual replicates.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove chlorine (pH-value: 6.0 - 8.5, Hardness 40 - 180 mg CaCO3 / L). The water is analysed once a year acc. to German tap water regulation.
- Culture medium different from test medium: no
- Intervals of water quality measurement:
pH-Value, temperature and oxygen saturation were measured in all vessels at the beginning of the test and every 24 h (from old and new media). Total hardness of the water was determined at start and any renewal from the control replicate. During the test the room temperature was recorded continuously with a thermohygrograph. Water quality parameters were determined to be within the acceptable limits. Equipement:
Oxygen: Oxygen meter "Oxi 197 - S", WTW
pH: Multilab 340i, WTW
Hardness: Spectrophotometer, CADAS 100 LPG 158, DR. LANGE
Thermohygrograph: LAMBRECHT, Göttingen

OTHER TEST CONDITIONS
- Adjustment of pH: No pH-related effects were found in the preliminary test, therefore no pH-adjustment was necessary for the definitive test.
- Photoperiod: A natural photoperiod occurred during the test.
- Light intensity: 0.1 -10 µmol/m^2*s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality: Observations were made after 2, 24, 48, 72 and 96 h.
- Symptoms of toxicity (visible abnormalities e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.)): see above

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 100 mg a.i./L (238 mg/L test item)
- Results used to determine the conditions for the definitive study: yes

TEST PERFORMANCE
Based on the results of a preliminary range finding test (semi-static conditions, NON-GLP), the definitive study was performed as a limit test with a concentration of 238 mg/L (100 mg a. i./L). One control with water (without test item) was tested under the same conditions as the test groups. In the test concentrations and control 7 fish were tested. The test fish were not fed during the test. After 2, 24, 48, 72 and 96 h mortality and behaviour of the test fish was observed. Measurements and observations were carried out as described above.

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

No abnormal behaviour of exposed fish was observed during 96h exposure period. Mortality did not occur neither in the treatment group nor the control group. Therefore, the derived 96h-LC50 was > 100 mg a.i./L.

With regard to the test solution, precipitation or any other observation were not reported. Recovery rates of the active ingredient were 80%. Therefore, effect concentrations are given on the basis of nominal concentrations.

VALIDITY CRITERIA
- O2-saturation was >= 60 % of the air saturation value
- Mortality in the control was 0 %

Reported statistics and error estimates:

no effects observed

Sublethal observations / clinical signs:

Cumulative Mortality [%] in the Test Vessels

Concentration [mg/L]	Test duration [h]
	2	24	48	72	96
238	0	0	0	0	0
Control	0	0	0	0	0

Observations in the Test Vessels

Concentration [mg/L]	Effect*	Test duration [h]
		2	24	48	72	96
238	(1)	7/7	7/7	7/7	7/7	7/7
Control	(1)	7/7	7/7	7/7	7/7	7/7

*) The numbers in brackets correspond to the following observations:

(1) Normal behaviour

Summary table DOC Analysis

Test item		Theoretical DOC	DOC [mg/L]				DOC [mg/L]
[mg test item/L]	[mg a.i./L]	[mg/L]	Parallel determination		Mean	RR [%]	Parallel determination		Mean	RR [%]
			0 h - fresh media
238	100	76.6	75.7	74.6	75.1	98
Control		-	0.1	0.2	0.1	-
			24 h (fresh media)				24 h (old media)
238	100	76.6	75.2	75.3	75.3	97	74.3	74.9	74.6	96
Control		-	1.2	1.2	1.2	-	0.8	0.9	0.8	-
			48 h (fresh media)				48 h (old media)
238	100	76.6	72	73.2	72.6	95	76.2	76.5	76.3	98
Control			0	0	0	-	1.4	1.3	1.3	-
			72 h (fresh media)				72 h (old media)
238	100	76.6	72.2	72.3	72.3	94	73.2	74	73.6	96
Control			0	0	0	-	0	0.1	0.1	-
							96 h (old media)
238	100						76.5	77.2	76.8	100
Control							0.1	0.3	0.2	-

RR = Recovery rate (calculated from mean value minus control)

Validity criteria fulfilled:

yes

Conclusions:

96h-LC50 (Danio rerio, mortality) > 100 mg a.i./L (nominal)

Executive summary:

The acute toxicity of the test substance to fish (Zebrafish) was determined according to OECD 203 (1992) and in compliance with GLP. A semi-static limit test with daily renewal of the test media was conducted with the limit concentration of nominal 100 mg a.i./L. The concentration of the test substance and the control were analytically verified via DOC (dissolved organic carbon). DOC was used as analytical method because HPLC was not applicable to detect the test item concentration applied in this study. The recovery rates of the test substance were close to the nominal concentrations (recovery rates 94-96 %), and therefore effect concentrations were based on nominal concentrations. During the 96 hours exposure period, no mortality occurred in neither treatment group nor control (n=7, 1 replicate each). The derived effect concentration was 96h-LC50 (Danio rerio, mortality) > 100 mg a.i./L (nominal). The test was considered reliable and adequate for the environmental hazard assessment for aquatic organisms. The validity criteria of the guideline were fulfilled.  

Description of key information

96h-LC50 (Danio rerio, mortality) > 100 mg a.i./L (nominal, OECD 203, 2002)

Additional information

A reliable key study is available on the acute toxicity of the submission substance (RL1). Acute toxicity of the test substance to fish (Zebrafish) was determined according to OECD 203 (1992) and in compliance with GLP.  A semi-static limit test with daily renewal of the test media was conducted with the limit concentration of nominal 100 mg a.i./L. The concentration of the test substance and the control were analytically verified via DOC (dissolved organic carbon). DOC was used as analytical method because HPLC was not applicable to detect the test item concentration applied in this study. The recovery rates of the test substance were close to the nominal concentrations (recovery rates 94-96 %), and therefore effect concentrations were based on nominal concentrations. During the 96 hours exposure period, no mortality occurred in neither treatment group nor control (n=7, 1 replicate each). The derived effect concentration was 96h-LC50 (Danio rerio, mortality) > 100 mg a.i./L (nominal). The test was considered reliable and adequate for the environmental hazard assessment for aquatic organisms. The validity criteria of the guideline were fulfilled.