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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 02 JUL 2002 to 04 JUL 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

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:

- All treatment groups and control
- Sampling: from freshly prepared media after 0 and 24h and from old media after 24 and 48h
- Sample storage conditions before analysis: not reported

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Stock solution: 238 mg/L, prepared with dilution water
- Dispersion treatment: Agitation
- Equipement:
Dispersion treatment: Rotating Shaker 3040, GFL, Centrifuge ZK 364, ZIRBUS

CONTROL (pH)
- Four replicates of the highest concentration level were tested as pH-control. The stock solution (pH-value = 9.39) was adjusted with 1 N HCL to pH 7.05 (0 h) and 7.04 (24 h).

CONTROL
- Dilution water without test item tested under the same conditions as the test groups.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna STRAUS (Clone 5)
- Source: Institut für Wasser-, Boden- und Lufthygiene des Bundesgesundheitsamtes, Berlin, FRG
- Feeding during test: no

ACCLIMATION
- Acclimation period: 2h before test start
- Acclimation conditions (same as test or not): yes
- Culture: In 2-3 L glass vessels with approximatively 1.8 L culture medium, at 21 °C (temperatures of 20-25 °C were tolerated), in an incubator
- Culture medium: Culture medium Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Type and amount of food: mix of Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 10^6 cells/mL.
- Feeding frequency: 5-7 time per week ad libitum
- Health during acclimation (any mortality observed): not reported

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
2 to 24 h old daphnids were used for the preliminary and definitive test. Daphnids had been obtained by removing the mother animals twice within 22 h.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

251 - 254 CaCO3 [mg/L] (min - max)

Test temperature:

20 ± 1 °C

pH:

Treatment groups: 7.77 - 9.39 (min - max)
Ph-control: 7.04 - 7.30 (min - max)

Dissolved oxygen:

7.77 - 8.58 mgO2/L (min - max)

Conductivity:

679 - 680 [µS/cm] (min - max)

Nominal and measured concentrations:

Nominal concentrations: 100 - 50 - 25 - 12.6 - 6.3 mg a.i./L (238 - 119 - 60 - 30 - 15 mg/L test item)
Measured concentrations: see any other information on results.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (5 cm ID x 8 cm H), 50 mL
- Fill volume: 20 mL
- Aeration: not reported
- Renewal rate of test solution: The water was renewed after 24 h.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per pH control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to EEC 92/69 L383A C.2 (Annex)
- Culture medium different from test medium: no
- Intervals of water quality measurement: Prior to test start pH-value, dissolved oxygen concentration, conductivity, total hardness and temperature of the dilution water were measured. In the fresh media (0 h and 24 h) the water parameters (pH value, oxygen concentration) were measured in one additional replicate per concentration and control. In the old media (24 h and 48 h) the water parameters were measured in all replicates per concentration and control. The room temperature was recorded throughout the test with a thermohygrograph.
- Equipement:
Oxygen: Oximeter, Oxi 530 (WTW)
pH value: pH-Meter inoLab pH Level 1 (WTVV)
Temperature: Hygro-thermograph (LuFFT)
Water hardness: Spectrophotometer, Cadas 100 LPG 158 (DR. LANGE)

OTHER TEST CONDITIONS
- Adjustment of pH: no, but pH controls were run in parallel: Four replicates of the highest concentration level were tested as pH-control. The stock solution (pH-value = 9.39) was adjusted with 1 N HCL to pH 7.05 (0 h) and 7.04 (24 h).
- Photoperiod: 16h illumination
- Light intensity: diffuse light, max. 20 µmol/m^2*s (corresponding to 1200 lx)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Immobility: in all groups after 24 h and 48 h
- Equipement:
Fluorescence of algae Chlorophyll-Fluorometer (KLEINFELD)

VEHICLE CONTROL PERFORMED: yes/no

RANGE-FINDING STUDY
- Test concentrations: 4 concentration levels of the test item ranging from 0.238 to 238 mg/L
- pH control: a pH-control (238 mg/L) was prepared because the pH-value of the stock solution was > 8.6.
- Spacing factor: 10
- Results used to determine the conditions for the definitive study: yes

TEST PERFORMANCE
The definitive study was performed with 5 concentration levels of the test item ranging from 15 to 238 mg/L set up in a geometric series with a factor of 2.0 under semi-static conditions to enable the determination of immobilization after 24 and 48 hours. In addition a pH-control (238 ring/L) was prepared. The water was renewed after 24 h. 2 to 24 h old daphnids were used for the preliminary and definitive test. They had been obtained by removing the mother animals twice within 22 h. The daphnids had been acclimatized to the dilution water 2 h before test started under test conditions. Control replicates without test item were tested under the same conditions as the test groups. In the preliminary and in the definitive test the immobilisation of the daphnids was observed after 24 and 48 hours of exposure. Water parameters were measured at begin and after 24 h (new media) and at 24 h and 48 h (old media).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: adjusted to pH 7

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: asjusted to pH 7

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

8.9 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

28.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: CI 95%: 20.6 - 39.5

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

A summary table of results is provided in 'any other information on results'. After 24 hours of exposure, immobilisation was only observed in the two highest treatment groups. After 48 hours, no effect of immobilisation was recorded in the lowest treatment group, the control and the pH-control (pH-neutralised treatment group containing 100 mg a.i./L). Observed immobilisation effects occurred in a dose-dependent manner. The derived 48h-EC50 was 28.6 mg a.i./ L (CI 95%: 20.6 - 39.5) and 48h-EC10 was 8.9 mg a.i./L. No effect was observed at 100 mg a.i./L, neutralised to pH 7. Therefore, 48h-EC50 with pH 7 was > 100 mg a.i./L. See justification in Overall remarks for using the adjusted test concentration as valid effect concentration.

The mean recovery rate for measure concentrations was > 80 %, except for the lowest concentration. Effect concentrations were based on nominal concentrations. The test item was clearly dissolved in all concentration levels throughout exposure. The pH-value varied by slightly more than 1.5 units in the treatment groups and laid in the exactable range of 7.04 - 9.39 (guideline indicates the pH should be in the range of 6 to 9). The pH increased with increasing test concentrations.

Validity cirteria
The validity criteria were fulfilled:
• In the control group no Daphnia were immobilised or trapped on the surface of the water.
• The dissolved O2 concentration was > 5.21 mg/L (60 % of the air saturation value at the temperature used).
• The EC50-value after 24 h of the reference item was in the range of 1.0 to 2.5 mg/L.

Results with reference substance (positive control):

- The reference item is routinely tested in order to check test system and test conditions (once per month).
- Test concentrations: 0.58 - 1.0 - 1.8 - 3.2 - 5.8 mg/L
- Range of validity: 1.0 mg/L < EC50 (24 h) < 2.5 mg/L

The percentage immobility for the reference item was determined after 24 h. The EC100 value was determined directly from the test results. The EC10 and EC50 with 95 % confidence interval (Cl) was determined in a probability network by interpolation according to standard procedures.

- EC- values after 24 h of the reference item in [mg/L]: EC10: 0.6; EC50: 1.4 (Cl 1.1-1.8); EC100: 3.2

The EC50 - value of reference item potassium dichromate after 24 h was within the prescribed concentration range of 1.0 - 2.5 mg/L of validity criteria according to AQS: DIN Guideline 38412 L 30.

Reported statistics and error estimates:

EC100-values were deduced directly from the dose-response-relationship after 24 and 48 h. There was no mathematical calculation. EC10 and EC50-values were calculated via probit analysis. Probits according to WEBER (1986). Calculation of the confidence intervals was carried out using standard procedures according to BREITIG & TÜMPLING (1982). Calculations were carried out using software SigmaPlot rel. 2000 (2000), SPSS CORPORATION.

BREITIG G. & TÜMPLING W. (1982): Methoden der Wasseruntersuchungen Bd.II, VEB Gustav Fischer Verlag, Jena
WEBER E. (1986): Grundriß der biologischen Statistik, 9. Auflage

Percentage of Daphnids Incapable of Swimming after 24 and 48 h of Exposure (n = 20)

Test item [mg/L]		Test duration [h]
		24					48
[mg test item/L]	[mg a.i./L]	1	2	3	4	MV	1	2	3	4	MV
238	100	0	0	0	20	5	80	80	60	60	70
119	50	0	20	0	0	5	60	100	60	80	75
60	25	0	0	0	0	0	40	20	60	40	40
30	12.6	0	0	0	0	0	20	40	0	20	20
15	6.3	0	0	0	0	0	0	0	0	0	0
238 (neutralized)	100 (neutralized)	0	0	0	0	0	0	0	0	0	0
Control		0	0	0	0	0	0	0	0	0	0

MV = Mean value

DOC - Analysis of New (0, 24 h) and Old Media (24, 48 h)

Test item		Theoretical DOC	DOC [mg/L]
			start				end
[mg test item/L]	[mg a.i./L]	[mg/L]	0 h	24 h	Mean	RR [%]	24 h	48 h	Mean	RR [%]
238	100	76.6	71.7	71.9	71.8	94	72	72	72	94
119	50	38.3	37	36.1	36.6	96	39.9	36	38	99
60	25	19.15	20.5	17.2	18.9	99	20	17	18.5	97
30	12.6	9.58	9	7.7	8.4	88	9.22	7.7	8.5	89
15	6.3	4.79	4.2	3	3.6	75	4.11	3	3.6	75
238 (neutralized)	100 (neutralized)	76.6	72.4	72.9	72.7	95	72	72.8	72.4	95
Control		-	0	0	0	0	0	0	0	-

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

Validity criteria fulfilled:

yes

Remarks:

(adopted 1984, 2004)

Conclusions:

No effect was observed at 100 mg a.i./L, neutralised to pH 7 and therefore the 48h-EC50, adjusted to pH 7 > 100 mg a.i./L.

Executive summary:

The acute immobilisation of the test substance was determined according to OECD 202 (1984) and in compliance with GLP. The study was conducted under semi-static conditions over a duration of 48 hours. Twenty test organisms were exposed in groups of five to each test concentration (nominal: 6.3 - 12.6 - 25 - 50 - 100 mg a.i./L), pH control (100 mg a.i./L, neutralised to pH7) and control. The test concentrations and control were analytically verified via analysis of dissolved organic carbon (DOC, acc. to DIN Guideline 28409, part 3). The mean recovery rate was 91 %.

After 24 hours of exposure, immobilisation was only observed in the two highest treatment groups. After 48 hours, no effect of immobilisation was recorded in the lowest treatment group, the control and the pH-control (pH-neutralised treatment group containing 100 mg a.i./L). Observed immobilisation effects occurred in a dose-dependent manner. The derived 48h-EC50 was 28.6 mg a.i./ L (CI 95%: 20.6 - 39.5) and 48h-EC10 was 8.9 mg a.i./L. No effect was observed at 100 mg a.i./L, neutralised to pH 7. The mean recovery rate for measured concentrations was > 80 %, except for the lowest test concentration. Effect concentrations were based on nominal concentrations (RL2).

The test item was clearly dissolved in all concentration levels throughout exposure. The pH-value varied by slightly more than 1.5 units in the treatment groups and was in the acceptable range of 7.04 - 9.39 (guideline indicates the pH should be in the range of 6 to 9), which is related to the alkaline characteristics of the test substance. In fact, the pH increased with increasing test concentrations. Therefore, observed effects of immobilisation are very likely linked to pH-effect rather than inherent toxicity of the test substance. The assumption is supported by the pH-control at 100 mg a.i./L adjusted to pH 7, where no immobilisation of daphnids occurred during 48 hours. According to OECD 23 and ECHA guidance R.7b, pH should be adjusted to lie within the specified range for the test using a suitable buffer, where the substance itself causes a change to the pH of the test medium. This is the case for the test substance. Therefore, the valid 48h-EC50 for the immobilisation of Daphnia magna was considered > 100 mg a.i./L.

The test was considered reliable and adequate for the environmental hazard assessment for aquatic organisms. The validity critera of the guideline were fulfilled (OECD 202 adopted 1984, 2004).  

Description of key information

No effect observed at 100 mg a.i./L, adjusted to pH 7; 48h-EC50 (Daphnia magna, immobilisation) > 100 mg a.i./ L (nominal) ( OECD 202, 2002)

Additional information

A reliable key study is available for the acute immobilisation of the test substance to Daphnia magna (RL1). Acute immobilisation of the test substance to Daphnia magna was determined according to OECD 202 (1984) and in compliance with GLP. The study was conducted under semi-static conditions over a duration of 48 hours. Twenty test organisms were exposed in groups of five to each test concentration (nominal: 6.3 - 12.6 - 25 - 50 - 100 mg a.i./L ), pH-control (100 mg a.i./L, adjusted to pH 7) and control. Test concentrations were analytically verified. After 48 hours, no effect of immobilisation was recorded in the lowest treatment group, the control and the pH-control. The derived 48h-EC50 was 28.6 mg a.i./ L (nominal) and 48h-EC10 was 8.9 mg a.i./L (nominal). No effect was observed at 100 mg a.i./L, neutralised to pH 7. The pH-value varied by slightly more than 1.5 units in the treatment groups and was in the acceptable range of 7.04 - 9.39 (guideline indicates the pH should be in the range of 6 to 9), which is related to the alkaline characteristics of the test substance. Observed effects of immobilisation are linked to pH-effect rather than inherent toxicity of the test substance. The assumption is supported by the pH control at 100 mg a.i./L and pH 7, where no immobilisation of daphnids occurred during 48 hours. According to OECD 23 and ECHA guidance R.7b, pH should be adjusted to lie within the specified range for the test using a suitable buffer, where the substance itself causes a change to the pH of the test medium. This is the case for the test substance. Therefore, the valid 48h-EC50 for the immobilisation of Daphnia magna was considered > 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.