1,6-Hexanediamine, N1,N6-bis(1,2,2-trimethylpropyl)-

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:

5 to 7 October 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Followed established guideline and GLP requirements

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: a 100 mg a.i./L primary stock solution prepared by placing 0.0513 of test article in 500 mL volumetric flask and bringing to volume with dilution water. Resulting solution clear and colorless with slight oily surfact after 10 minutes mixing with magnetic stir plate. pH adjusted to 7.1 by adding 0.2 mL of 5.0 N hydrochloric acid solution. Test solutions then prepared from dilutions of the water accommodated fraction of the primary stock solution. After mixing for 30 seconds with a glass rod, all test solutions clear and colorless with no visible undissolved test substance. Each test solution divided among 4 replicate test vessels.
- Differential loading: Daphnids impartially selected and distributed to intermediate vessels, two at a time, until each vessel contained 2 organisms. Procedure repeated until each vessel contained 5 daphnids. Test began when daphnids in each intermediate vessel were added to the concentration replicates and control replicates.
- Controls: dilution water prepared from fortified well water
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None observed in test solutions

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water Flea
- Strain: Daphnia magna
- Source: Springborn Smithers culture facility
- Age at study initiation: < 24 hours
- Feeding during test: no


ACCLIMATION
- Acclimation period: daphnids less than 24 hours old at start of test
- Type and amount of food: unicellular green algae (Ankistrodesmus falcatus) and a suspension of YCT (yeast, cereal leaves, and flaked fish food) while in culture
- Feeding frequency: daily except during testing

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Culture water was prepared by fortifying well water based on formula for hard water (US EPA 1975) and filtering it through an Amberlite XAD-7 resin column to remove any potential organic contaminants. Total hardness and alkalinity ranges as CaCO3 of 150 to 160 mg/L an d96 to 100 mg/L respectively. pH of the culture water had a pH range of 7.8 to 8.2. Test water was prepared by diluting culture water with deionized water and mixing.

Test temperature:

20 to 21 degrees C

pH:

pH of dilution water 8.0. See tables in materials and methods for pH during test

Dissolved oxygen:

See table in materials and methods

Nominal and measured concentrations:

Nominal concentrations: 0, 0.010, 0.026, 0.064, 0.16, 0.40, and 1.0 % water accommodating fraction.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass beakers
- Material, size, headspace, fill volume: 250 ml glass beakers filled with 200 mL test solution and placed into temperature control water bath

- No. of organisms per vessel: 5 per replicate vessel
- No. of vessels per concentration (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water prepared by diluting fortified well water with deionized water and mixing thoroughly.
- Total organic carbon: TOC of dilution water source ranged from 0.32 to 0.49 mg/L in 6 month period prior to test. Prior to test initiation, 3 35-ml samples of dilution water used for test were analyzed for TOC, and found to have a range of 0.37 to 0.41 mg/L.
- Particulate matter: None observed
- Metals: Dilution water source samples analyzed regularly for toxic metals.
- Pesticides: Dilution water source samples analyzed regularly for pesticides
- Alkalinity: Dilution water: 140 mg/L as CaCO3 hardness. Total alkalinity 86 mg/L as CaCO3.
- Conductivity: Specific conductivity 500 umhos/cm
- Culture medium different from test medium: Culture medium was fortified well water; test medium was fortified well water diluted with deionized water.
- Intervals of water quality measurement: Monthly for TOC, regularly for pesticides, PCBs, and toxic metals.

OTHER TEST CONDITIONS
- Adjustment of pH: Primary stock solution adjusted to pH 7.1 before test concentrations prepared
- Photoperiod: 16 hours light, 8 hours dark
- Light intensity: 68 to 83 footcandles at surface of media provided by fluorescent bulbs.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilization, lethargy, and presence on surface of test solution noted during observation at 24 or 48 hours.

TEST CONCENTRATIONS
- Range finding study: yes, two replicates using 5 daphnids per treatment level and control (dilution water)
- Test concentrations: 0, 0.010, 0.10, 1.0, 10, and 100 mg/L nominal concentrations
- Results used to determine the conditions for the definitive study: 100% immobilization seen at 24 hours with concentrations 1.0 mg/L and higher. At 48 hours, no immobilization or adverse effects were seen in control, or in the lower concentrations (0.010 and 0.10 mg/L.) Due to the nature of the test material, concentrations chosen for the definitive test were to be per cents of a Water accommodating fraction of a 100 mg a.i./L test solution.

Reference substance (positive control):

yes

Remarks:

potassium dichromate tested twice yearly in a 24 hour reference test

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 1 other: % water accommodating fraction

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: not possible to calculate confidence limits

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.4 other: % water accommodating fraction

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence intervals 0.16 to 1.0 % WAF

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.064 other: % Water accommodating fraction

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

behaviour

Results with reference substance (positive control):

- Results with reference substance valid? Yes, but not concurrent with test
- EC50/LC50: 24 hour EC50 for potassium dichromate conducted from 15 to 16 March 2007 was 3.3 mg/L. This value was within the expectations for the test organisms.

Reported statistics and error estimates:

Nominal concentrations tested and the corresponding immobilization data from the definitive test were used to estimate the median effect concentrations (EC50) and 95% confidence intervals. The EC50 was defined as the concentration of the test substance in dilution water which caused immobilization of 50% of the test organism population at the stated time interval. If at least one test concentration caused immobilization equal or greater than 50%, then a computer program (Stephan, 1982) was used to calculate the EC50 and 95% CL values. Three statistical methods were available in the program: moving average angle analysis, probit analysis and binomial probability. Moving average angle and probit analysis yield sound results only if at least 2 concentrations cause immobilization between 0 and 100%. Selection criteria for reported EC50 values included establishment of dose-response relationship, number of concentrations causing partial effects, and span of responses bracketing the EC50. If two or more methods produced acceptable results, the method with smallest 95% CL was chosen. NOEC for the 48 hour exposure was also determined.

Daphnia Immobilization and Observations

	24 hour Observation					48 hour Observation
Nominal Conc. (%WAF) a	A	B	C	D	Mean	A	B	C	D	Mean
	Cumulative Percent of Immobilized Organisms b
Control	0 (0)	0 (0)	0 (0)	0 (0)	0	0 (0)	0 (0)	0 (0)	0 (0)	0
0.010	0 (0)	0 (0)	0 (0)	0 (0)	0	0 (0)	0 (0)	0 (0)	0 (0)	0
0.026	0 (0)	0 (0)	0 (0)	0 (0)	0	0 (0)	0 (0)	0 (0)	0 (0)	0
0.064	0 (0)	0 (0)	0 (0)	0 (0)	0	0 (0)	0 (0)	0 (0)	0 (0)	0
0.16	0 (0)	0 (0)	0 (0)	0 (0)	0	0 (0)	0 (0)	0 (0)	0 (0)	0 c
0.40	0 (0)	0 (0)	0 (0)	0 (0)	0	40 (2)	40 (2)	60 (3)	60 (3)	50 d
1.0	0 (0) d	0 (0) d	0 (0) e	0 (0) d	0	100 (5)	100 (5)	100 (5)	100 (5)	100

a Exposure solutions prepared from a 100 mg a.i./L test solution , presented as % of WAF

b Actual number of immobilized daphnia in parenthesis

c Several daphnia observed to be lethargic

d All surviving daphnia were observed to be lethargic

e One daphnid lethargic, and 4 daphnids on surface of test solution

EC50 values and 95% Confidence intervals and NOEC through 48 hours

		95% Confidence Intervals
Time period	EC50 (% WAF) a	Lower (% WAF)	Upper (% WAF)
24 hour	> 1.0	NA b	NA
48 hour c	0.40	0.16	1.0
	NOEC through 48 hours = 0.064% WAF

a  Concentrations listed are nominal

b  NA = Not applicable  EC50 value was empirically estimated, therefore, the 95% confidence intervals could not be calculated

c EC50 values and 95% confidence intervals were estimated by binomial probability

Validity criteria fulfilled:

yes

Conclusions:

In a static acute toxicity test to Daphnia magna, the 48 hour EC50 of the test article, based on nominal concentrations, was 0.40% of the water accommodating fraction, with 95% confidence limits of 0.16 to 1.0% WAF. The No-Observed-Effect Concentration (NOEC) was determined to be 0.064% WAF. The EC50 was estimated by binomial probability. The water accommodating fraction was prepared from a 100 mg a.i./L test solution.
Based on the water solubility of the test substance of 86.4 mg/L as determined in the water solubility test, the EC50 is approximately 0.34 mg/L and the NOEC is 0.05 mg/L. The apparent high toxicity to Daphnia is thought to be due to the reduction in surface tension of the water and thus rather an indirect physical effect than direct toxicity. Furthermore as shown in the study on acute toxicity to fish, in nature the substance will bind to humic acids and is thus unlikely to reduce the surface tension of natural waters. This study will therefore not be considered for classification and risk assessment purposes.

Executive summary:

In a static acute toxicity test to Daphnia magna, the 48 hour EC50 of the test article, based on nominal concentrations, was 0.40% of the water accommodating fraction, with 95% confidence limits of 0.16 to 1.0% WAF. The No-Observed-Effect Concentration (NOEC) was determined to be 0.064% WAF. The EC50 was estimated by binomial probability. The water accommodating fraction was prepared from a 100 mg a.i./L test solution.

Based on the water solubility of the test substance of 86.4 mg/L as determined in the water solubility test, the EC50 is approximately 0.34 mg/L and the NOEC is 0.05 mg/L. The high toxicity to Daphnia could be due to the reduction in surface tension of the water and thus rather an indirect physical-chemical effect than direct toxicity.

Description of key information

An acute toxicity test on aquatic invertebrates has been performed. The test was perfomed in a static conditions with Daphnia magna during 48 hours using the EU method 2 under GLP conditions. Due to the limited solubility of the test material in the test medium, the study was performed using a water accomodated fraction. Based on the water solubility of the test substance of 86.4 mg/L as determined in the water solubility test, the EC50 is approximately 0.34 mg/L and the NOEC is 0.05 mg/L. The apparent  high toxicity to Daphnia is thought to  be due to the reduction in surface tension of the water and thus rather an indirect physical effect than direct toxicity. Furthermore as shown in the study on acute toxicity to fish, in nature the substance will bind to humic acids and is thus unlikely to reduce the surface tension of natural waters. This study will therefore not be considered for classification and risk assessment purposes. 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.34 mg/L