2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-18 to 2015-07-24

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

adopted July 26, 2013

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Nominal concentrations of 0.256- 0.64 - 1.60 - 4.0 - 10.0 mg/L (factor: 2.5)
- Arithmetical mean measured concentrations of 0.322 - 0.817 - 1.75 - 4.40 - 10.9 mg/L
- Samples of test media including the control group were taken from alternating test replicates on days -1, 0 and weekly thereafter until the end of
exposure.
- Stock solutions were sampled and analysed from freshly prepared and aged solutions of one application interval.
- To prove the stability over at least three days, the old stock solutions (age: 3 days) were analysed after exchange of the syringes

Vehicle:

no

Details on test solutions:

- No organic solvent was used. The stock solutions were prepared in demineralised water.
- A flow-through exposure design was carried out.
- Membrane piston pumps provided the water flow-through in two consecutively subsequent orders.
- The first order of membrane piston pumps delivered the dilution water in 5 separate streams. A tubing pump was used for the application of stock
solutions with different concentrations.
- The stock solutions and the dilution water were mixed in a mixing chamber (approx. Vol. 1.5 L) by magnetic stirring.
- A second row of membrane piston pumps provided the test solutions from these mixing chambers to the test aquaria (approx. Vol. 7.6 L;
four replicates per test concentration and control), where the fish were exposed.
- The accuracy of the water flow-through was checked prior to the start of exposure and daily on working days after test start.
- Water exchange in the mixing chamber was about 230 times per day (14.375 L/h) and about 10 times per day in the test aquaria (3.17 L/h),
respectively. An equilibration period of 7 days was carried out prior to exposure.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Danio rerio (zebrafish), Gnathostoma, Pisces, Osteichthyes, Teleostei, Cypriniformes, Cyprinidae
- Source: All fish used in the test were gained at DR.U.NOACK-LABORATORIEN from a single brood stock (supplier: Umweltbundesamt,
Schichauweg 58, D-12307 Berlin, Germany)
- Age at study initiation (mean and range, SD): eggs
- Method of breeding: unexposed, mature zebrafish with an age of 10 months was used for the egg production
- Maintenance of brood fish:Spawners were maintained in aquaria with a loading capacity of a minimum of 1 L water per fish.
- Temperature: 25 ± 2 °C
- Dissolved oxygen concentration > 60 % of air saturation value
- pH value: 6 – 8.5
- Photoperiod: 16 h light / 8 h dark cycle
(2 transition periods, 30 minutes each)
- Diffuse light (0.1 - 10 µmol photons · m-2 · s-1 on water surface)
- Food: Artemia salina nauplii, 48 hours old, ad libitum; Daphnia magna, juvenile and adult daphnids, ad libitum;
dry food sera vipan SERA, ad libitum.
- No disease treatments were administered.
- Feeding of test fish: Feeding started 2 days after hatch (on post-hatch day 1).
Larvae were first fed with starter food (e. g. TetraMin Baby Hauptfutter, TETRA GMBH, D-49304 MELLE, Germany).


ACCLIMATION
- Acclimation period: eggs were taken, washed in dilution water and transferred immediately into vessels containing the respective exposure
solutions,

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 d

Post exposure observation period:

none

Hardness:

Total hardness: 10 - 250 mg CaCO3/L

Test temperature:

Water temperature: 26 ± 1.5 °C

pH:

pH-value: 6.0 - 8.5

Dissolved oxygen:

Not less than 60 % of air saturation value

Salinity:

not measured

Nominal and measured concentrations:

- Nominal concentrations of 0.256- 0.64 - 1.60 - 4.0 - 10.0 mg/L (factor: 2.5)
- Arithmetical mean measured concentrations of 0.322 - 0.817 - 1.75 - 4.40 - 10.9 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria of 8.7 L provided with mesh coated fittings allowing flow-through of test media (dimensions: 22/22/18 cm) were used.
The volume of the test media was approximately 7.6 L. A randomised block design with each treatment being present in each block was established.
- Aeration: No additional aeration of the test vessels was provided. The dilution water supply tank was aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): Membrane piston pumps provided the water flow-through in two consecutively
subsequent orders. The first order of membrane piston pumps delivered the dilution water in 5 separate streams. A tubing pump was used for the application of stock solutions with different concentrations.
- Renewal rate of test solution (frequency/flow rate):The accuracy of the water flow-through was checked prior to the start of exposure and daily on working days after test start. Water exchange in the mixing chamber was about 230 times per day (14.375 L/h) and about 10 times per day in the
test aquaria (3.17 L/h), respectively. An equilibration period of 7 days was carried out prior to exposure.
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): not required
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: A loading rate not exceeding 0.5 g/L wet weight fish per 24 hours and not exceeding 5 g/L of solution at any time was
maintained.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding. The water was filtered on activated charcoal and aerated for at
least 24 h to remove chlorine.
- Total organic carbon: 10 - 250 mg CaCO3/L
- Metals: filtered on activated charcoal
- Pesticides: filtered on activated charcoal
- Chlorine: filtered on activated charcoal
- Alkalinity: filtered on activated charcoal
- Ca/mg ratio:
- Conductivity:
- Salinity:
- Intervals of water quality measurement:
Continuously: - Temperature in the dilution water (once per hour).
At least 3 times per week: - Dissolved oxygen and temperature in one replicate of each test group
- Check of flow rates of the test media (variation < 10 % throughout exposure)
Weekly: - pH-value in alternate replicates of each test group
- TOC and Chlorine from dilution water
- Total hardness in one replicate of the control and the highest test item concentration


OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: A daily 16 / 8 h photoperiod (light / dark) was maintained throughout exposure.
- Light intensity: 0.1 - 10 µmol photons · m-2 · s-1 (corresponding to approximately 7 – 750 Lux).


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality: Criteria for mortality vary according to life stage:
For eggs/embryos: If fungus growth on eggs was observed, these eggs were removed and counted. 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 and change in coloration was checked daily. Mortality caused by absence of heartbeat was checked, if applicable. Dead
eggs/embryos were discarded.
For larvae and juvenile fish: Immobility and/or lack of reaction to mechanical stimulus. Dead larvae or juvenile fish were discarded.

- Further effects:
Abnormal appearance and behaviour were also recorded at adequate intervals.
The number of larvae or fish showing abnormality of body form 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 were
recorded by visually inspecting each replicate.
- Measurement of fish size:
At the end of exposure (post-hatch day 30) the fish were euthanized in a Benzocaine solution and the individual total length of all survivors was
measured to the nearest 0.5 mm.
- Measurement of fish wet weight:
At the end of exposure (post-hatch day 30) all surviving fish were weighed on replicate basis. Fish were blotted on paper towels to remove excess
moisture prior to weighing. The mean wet weight per animal was calculated from the number of surviving fish.


VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Preliminary range finding test was conducted over 6 days under flow-through conditions (n = 40 per treatment group with 4 replicates and 10 eggs each under flow-through conditions)
- Test concentrations: 1 mg/L, 10 mg/L
- Results used to determine the conditions for the definitive study: yes

POST-HATCH DETAILS
- Begin of post-hatch period: Per definition the post-hatch period begun when at least 90 % (better 95 %) of all fertilized and living embryos in the
control groups had hatched (about day 5 ± 2 of the study). On study day 4, 96 % of the control larvae had hatched. Therefore, study day 4 was
defined as post-hatch day 0 (= PHD 0).
- No. of hatched eggs (alevins)/treatment released to the test chamber: The number of hatched larvae was determined daily until post-hatch day 1.


FERTILIZATION SUCCESS STUDY
- Number of eggs used:
- Removal of eggs to check the embryonic development on day no.:

Reference substance (positive control):

not required

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

>= 10.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

30 d

Dose descriptor:

LOEC

Effect conc.:

> 10.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Mortality/survival at embryo, larval, juvenile, and adult stages: The post-hatch survival in the control replicates met the validity criteria of the
guideline (required: > 75 %). The fry survival (post-hatch survival) at the end of the study was 89 % in the control group. The post-hatch survival in
the arithmetic mean calculated test concentrations 0.322 to 10.9 mg/L was 77 to 85 %

- Days to hatch or time to release of young: Hatch began on study day 3 in the control and all test item concentrations and continued until study day 6. Study day 4 was determined to be post hatch day 0 (PHD 0) with a control hatching rate of 96 %.

- Numbers hatched, Numbers of offspring produced, or Number of offspring per live female per day: The egg fertilization rate, determined on study
day 0 (start of the exposure) was 98 %.

- Number of fish in swim-up stage at one or more time periods (e.g., day x1, x2): Swim-up was observed for a 4-day period from study days 4 to 7
(see Table 6). Newly hatched fry began to swim up on study day 4 (PHD 0). On study day 5 (PHD 1), all surviving larvae had swum up.
No statistical analysis of swim-up data was carried out.

- Observations on body length and weight of young and/or exposed parents at one or more time periods: Fry growth, expressed as length and wet
weight, was measured on study day 34 (PHD 30) from all survivors. The statistical analysis of the data of all arithmetic mean calculated test
concentrations showed no significant differences for any parameter

- Number of healthy fish at end of test: see above
- Type of and number with morphological abnormalities: No biologically significant morphological effects were observed in any treatment.
- Type of and number with behavioural abnormalities: none
- Type and number of developmental effects: none
- Other biological observations: none
- Effect concentrations exceeding solubility of substance in test medium: none
- Incidents in the course of the test which might have influenced the results: none

Tables see below " Overall remarks, attachments"

Results with reference substance (positive control):

- Results with reference substance valid? not required
- Relevant effect levels:
- Other:

  Measured Concentrations in the Test Vessels during Exposure

             Measured Concentrations, Percent of Nominal Concentrations and Arithmetical Mean of test item in the Test Vessels during Exposure

             (Control, 0.256 and 0.640 mg/L)

Nominal test itemconcentration [mg/L]		Control	0.256		0.640
Sampling day [d]	Sampling date / Start of Analysis
		Meas. conc. [mg/L]	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
0	2015-06-18	< LOQ	0.309	121	0.716	112
6	2015-06-24	< LOQ	0.304	119	0.750	117
13	2015-07-01	< LOQ	0.445^1)	174	1.27^1)	198
19	2015-07-07	< LOQ	0.289	113	0.730	114
27	2015-07-15	< LOQ	0.305	119	0.724	113
33	2015-07-21	< LOQ	0.282	110	0.711	111
Arithmetical mean (d0 – 33) [mg/L]		< LOQ	0.322		0.817

             Measured Concentrations, Percent of Nominal Concentrations and Arithmetical Mean of test item in the Test Vessels during Exposure

             (1.60, 4.00 and 10.0 mg/L)

Nominal test itemconcentration [mg/L]		1.60		4.00		10.0
Sampling day [d]	Sampling date / Start of Analysis
		Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
0	2015-06-18	1.31	82	3.33	83	8.68	87
6	2015-06-24	1.79	112	4.78	120	11.6	116
13	2015-07-01	1.76	110	4.36	109	11.0	110
19	2015-07-07	1.89	118	4.77	119	11.5	115
27	2015-07-15	1.88	118	4.56	114	11.3	113
33	2015-07-21	1.86	116	4.59	115	11.6	116
Arithmetical mean (d0 – 33) [mg/L]		1.75		4.40		10.9

Meas. Conc.                  =  Measured concentration (dilution factor taken into account)

                       %                            =  Percent of nominal concentration of the test item

                      LOQ                        =  Limit of quantification of theanalytical method(0.025 mg/L of the test item)

 

Validity criteria fulfilled:

yes

Conclusions:

The test item caused no significant effects on Zebrafish in an early life stage test, 30 days post hatch.
Based on the parameters hatch, growth (expressed as length and dry weight) and post-hatch survival (mortality) the overall NOEC (nominal,
post hatch day 0 – 30) was ≥ 10.0 mg/L, corresponding to 10.9 mg/L (arithmetical mean). The overall LOEC (nominal, post hatch day 0 – 30) was
> 10.0 mg/L, corresponding to > 10.9 mg/L (arithmetical mean).
For further NOEC and LOEC values. All effect values are given based on the arithmetical mean calculated concentrations of test item.

Executive summary:

Purpose of the study was, determination of the toxicity of test item to the early-life stages of the zebrafish acc. to OECD 210. The test was started by placing fertilised eggs into the test vessels and it lasted 34 days (30 days post-hatch). Lethal and sublethal effects (hatch, fry growth (expressed as length and weight) and post-hatch survival) were assessed and compared with control values to determine the LOEC, the NOEC and the EC_x-values.
A test was conducted under flow-through conditions with the nominal test item concentrations of 0.256 - 0.64 - 1.60 - 4.0 - 10.0 mg/L, corresponding to arithmetical mean measured concentrations of 0.322 - 0.817 - 1.75 - 4.40 - 10.9 mg/L.

The test was started by placing fertilized eggs into the test vessels and it lasted 34 days (30 days post-hatch). 80 eggs of Danio rerio were exposed to each test concentration and the control (4 replicates with 20 eggs each).

 

Different toxicological endpoints were determined: hatch, time to hatch, fry growth (expressed as length and fresh weight), morphological and behavioural effects and post-hatch survival. Specific analysis of various concentrations of test item in the test media and the control was carried out via LC-MS/MS. The test media were sampled and analysed prior to exposure on day -1 and during the exposure on study days 0, 6, 13, 19, 27 and 35. The measured concentrations of the test media during the exposure were in the range of 82 % to 198 % of the nominal values.

The test substance caused no significant effects on Zebrafish in an early life stage test, 30 days post hatch.

Based on the parameters hatch, growth (expressed as length and dry weight) and post-hatch survival (mortality) the overall NOEC (nominal, post hatch day 0 – 30) was≥10.0 mg/L, corresponding to 10.9 mg/L (arithmetical mean). The overall LOEC (nominal, post hatch day 0 – 30) was > 10.0 mg/L, corresponding to > 10.9 mg/L (arithmetical mean).

Description of key information

The long-term effects on Danio rerio were studied in a fish early life stage test according to OECD 210. The test substance caused no significant effects on Zebrafish, 30 days post hatch. Based on the parameters hatch, growth (expressed as length and dry weight) and post-hatch survival (mortality) the overall NOEC (nominal, post hatch day 0  30) was  10.0 mg/L, corresponding to 10.9 mg/L (arithmetical mean). The overall LOEC (nominal, post hatch day 0  30) was > 10.0 mg/L, corresponding to > 10.9 mg/L (arithmetical mean). 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

10 mg/L