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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-10 to 2015-07-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP standard

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

(2012)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

DETERMINATION OF TEST ITEM
- All concentration levels and the control were analysed once within 7 days at the start of an exposure interval (0 hours) and at the end of an
exposure interval (48 or 72 hours).

SAMPLING FOR THE ANALYTICAL MONITORING
- At the start of the exposure intervals, sampling was carried out after preparation of all test item concentrations.
- At the end of the exposure intervals (48 or 72 hours), samples were taken from additional replicates without daphnids and food algae.
- The additional replicates were incubated under test conditions until sampling.
- For the longest exposure interval of 72 hours samples were taken at the start (0 hours) and at the end of the exposure interval (72 hours) once
within the test period.

Vehicle:

no

Details on test solutions:

- Stock solution: A stock solution (40.0 mg/L of the test item were weighed out) was freshly prepared with dilution water for each exposure interval.
- Treatment: The stock solution was mixed thoroughly by inverting the measuring flask at least 10 times by hand. The clear stock solution was used
as highest test concentration and for preparation of the further test concentrations.
- Test concentrations / solutions: 5 test item concentrations in a geometric series with a separation factor of 2.5, prepared by diluting the stock
solution of 40.0 mg/L with the dilution water, were tested as follows: 1.02 - 2.56 - 6.40 - 16.0 - 40.0 mg/L.
- Control: 10 replicates of dilution water without test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Test system: Daphnia magna STRAUS (Clone 5)
- Reason for the selection of the test system: Daphnia magna is the preferred species in accordance with the test guideline and is bred at the test
facilityThe Daphnia magna culture of Clone 5 has shown to meet the validity criteria for the reproduction test of a mean of ≥ 60 living juveniles per
surviving parent animal when cultured under conditions described.
- Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
- Breeder: DR.U.NOACK-LABORATORIEN, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
- Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2°C, in an incubator, 16 h illumination;
light intensity of max. 20 µExm-2 x s-1
- Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, is used.
- Age of the daphnids at the start of the exposure: Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels latest 24 hours before the start of the exposure and discarded. The juveniles born within this period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.
- Culture feeding: The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella
subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae are cultured at the test facility.
- Origin of the food algae: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

see Table "Water Quality Parameters" in section "Overall remaks".

The total hardness should be above 140 mg/L CaCO3.

Test temperature:

see Table "Water Quality Parameters" in section "Overall remaks".

pH:

see Table "Water Quality Parameters" in section "Overall remaks".

The pH-values should be in the range of 6 - 9. Normally deviations between initial and final pH values should be < 1.5 units.

Dissolved oxygen:

see Table "Water Quality Parameters" in section "Overall remaks".

The oxygen concentration should be > 3 mg/L at the start and during the test period.

Salinity:

no data

Nominal and measured concentrations:

Nominal concentrations of the test item were selected based on the results of a non GLP preliminary test, conducted at the test facility as an acute
immobilisation test under static conditions over a period of 48 hours. Nominal test item concentrations: 1.02 - 2.56 - 6.40 - 16.0 - 40.0 mg/L.
The measured concentrations of the test item at the start of the exposure intervals (0 hours) were in the range of 97 to 110 % of the nominal values.
At the end of the exposure intervals (48 or 72 hours, respectively), the measured concentration of the test item were in the range of 95 to 118 % of
the nominal values

Details on test conditions:

- Test method: The study was performed with a semi-static test design. A medium renewal 3 times per week (i.e. on Monday, Wednesday and Friday)
was found to be suitable, because the test item was assumed to be stable under test conditions, which was proven in the non GLP preliminary range finding test resulting in stable test item concentrations over a period of 48 and 72 hours.
- Renewal of the test solutions: The test solutions were renewed 3 times per week (after 48 or 72 hours). For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette.
- Test duration: 21 days
- Test vessels: Glass beakers (5 (ID) x 8 (H) cm), 100 mL capacity, loosely covered with watch glasses
- Test volume: 50 mL
- Dilution water: Same as culture medium, therefore no acclimation necessary
- Number of daphnids and replicates: 10 daphnids each were used for all concentration levels and the control and individually held.
- Age of the daphnids at the start of the exposure: Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels latest 24 hours before the start of the exposure and discarded. The juveniles born within this period of
max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.
- Application: Per replicate, 50 g test solution were weighed out into the test vessels. This corresponds to 50 mL. The daphnids were transferred in a small amount of dilution water or test solution by pipette.
- Feeding: Daily feeding per test vessel with Pseudokirchneriella subcapitata (0.435 - 0.802 mL) and Desmodesmus subspicatus (0.624 - 1.90 mL)
suspension was provided as food corresponding to 0.2 mg C per Daphnia and day.
Variation was according to the density of the algae suspension, but was the same for all test groups on each feeding day.
- Test temperature (target): 20 ± 1 °C
- Light intensity (target): Max. 20 µE x m-2 x s-1
- Photoperiod: 16/8 h light/dark cycle
- Aeration: Test vessels were not aerated during the test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate p.a. (SIGMA)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

1.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.02 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

All Tables see "other informations on results incl. tables" and "remarks on results"

Adult Mortality
The test item induced significant adult mortality in the concentration levels of 2.56 mg/L (40 %), 6.40 mg/L (40 %), 16.0 mg/L (100 %) and
40.0 mg/L (100 %) during the test period of 21 days. In the concentration level of 1.02 mg/L, an adult mortality of 10 % was observed, which is not
biologically significant (see validity criteria for the control). In the control, all parental daphnids survived until the end of the test after 21 days.
The adult mortality was the most sensitive effect in this study. Based on the significant adult mortality at the concentration levels of 2.56 to
40.0 mg/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 1.02 mg/L and the
Lowest Observed Effect Concentration (LOEC) was assessed at 2.56 mg/L.
Based on the nominal test item concentrations, the EC10 was observed at 1.02 mg/L. The EC50 was calculated by sigmoidal
dose-response regression to be 5.26 mg/L (95% confidence limits: 2.23 – 11.8 mg/L). See Table 5

Reproductive Output
The number of juveniles in all replicates of the control and the test groups was counted and recorded every day. Results concerning the number of
living juveniles of all concentration levels and the control for every test day are presented in Table 14 to Table 19.
The number of living juveniles produced in the 10 replicates of all concentration levels and the control between the 8th and 21st day are presented in Table 6.
The average number of living juveniles per surviving parental daphnid at the end of the test after 21 days was 99 in the control group. The reproductive output of the surviving parental daphnids was not statistically significant reduced at any of the tested concentration levels of 1.02 to 6.40 mg/L in comparison to the reproductive output in the control (KRUSKAL-WALLIS One Way Analysis of Variance on Ranks, P = 0.665). Due to the lack of significant effects, no EC-values could be calculated.
The coefficient of variation of the number of living offspring produced per parental daphnid was 16 % in the control. At the concentration levels of 1.02 to 6.40 mg/L, the coefficient of variation was in the range of 13 to 14 % (Table 1 and Table 6). Generally in a well-run test, the coefficient of variation around the mean number of living offspring per parent animal should be ≤ 25 %. At the concentration levels 16.0 and 40.0 mg/L, no living offspring were observed and therefore no coefficient of variation could be calculated.

Intrinsic Rates of Natural Increase (IR)
The intrinsic rates of natural increase (IR) of the surviving parental daphnids accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The intrinsic rates of natural increase determined for the surviving daphnids at the concentration levels of 1.02 to 6.40 mg/L were comparable to the IR at the control (One Way Analysis of Variance,P = 0.075).For details, seeTable1,Table7.

Number of Broods and first Appearance of living Juveniles
At the end of the test after 21 days, the number of broods with living juveniles produced by the surviving parental daphnids was 4 - 5 in the tested
concentration levels of 1.02 to 6.40 mg/L and in the control. At the concentration levels of 16.0 and 40.0 mg/L all daphnids died before producing
juveniles.
The first appearance of living juveniles was observed on days 8 or 9 by all parental daphnids of the control and the concentration levels of
1.02 to 6.40 mg/L. For details, see Table 1 and Table 8.

Stillborn Juveniles and Aborted Eggs
The number of stillborn juveniles and aborted eggs per surviving parental daphnid of the test and control groups are presented in Table 9.
The fraction of dead juveniles (sum of stillborn juveniles and aborted eggs) expressed as a percentage, compared to the total number of produced
juveniles (alive, stillborn juveniles and aborted eggs) of the surviving daphnids is presented in Table 10.
No stillborn juveniles or aborted eggs were observed in the control and in the concentration level of 1.02 mg/L during the exposure period of
21 days. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was < 1 % at the concentration levels of
2.56 and 6.40 mg/L. At the concentration levels of 16.0 and 40.0 mg/L all daphnids died before producing juveniles.

Growth (Total Body Length and Dry Weight) of the Parental Daphnids
The mean values of the body length of the surviving parental daphnids in the tested concentration levels of 1.02 to 6.40 mg/L were determined to
be in the range of 4.92 to 4.97 mm per daphnid and 4.98 mm per daphnid in the control group.
The mean dry body weight of the surviving parental daphnids was determined to be in the range of 0.81 to 0.92 mg per daphnid in the concentration
levels of 1.02 to 6.40 mg/L and 0.78 mg per daphnid in the control (Table 1 and Table 11).

Presence of Males
No males were observed in the control or in the test groups during the test.

Occurrence of Ephippia (Winter Eggs)
No ephippia were observed in the control or in the test groups during the test

Results with reference substance (positive control):

In order to prove the validity of the test system and test conditions at the test facility, an acute immobilisation test according to AQS P 9/2 (02/2000) and OECD 202 (2004) is monthly carried out with potassium dichromate as reference item.
The percentage immobility for the reference item was determined after 24 hours. The EC50-value with 95 % confidence limits (Cl) was calculated by
sigmoidal dose-response regression. The EC50-value for the most recent of the monthly performed reference tests was:
EC50 : 2.00 mg/L (CI: 1.76 - 2.27 mg/L)
The EC50-value of the reference item potassium dichromate after 24 hours is within the prescribed concentration range of 0.6 - 2.4 mg/L of quality
criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium.
The EC50-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

Table 5: Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure  (n = 10)

Nominal test item concentration [mg/L]	Adult Mortality [%]
	7 days	14 days	21 days
40.0	100*	100*	100*
16.0	50*	100*	100*
6.40	0	0	40*
2.56	0	10	40*
1.02	0	10	10
Control	0	0	0

*   = biologically significant adult mortality observed (> 20 %)

Table 6: Number of living Juveniles in the Control and Test Groups after 21 Days

Nominal test item conc.   [mg/L]												No. of parents alive	Mean no. of juveniles per parent producing juveniles			Comparison versus control
	Number of living juveniles in replicate no.										Total no.				CV
																Red.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV±SD		[%]	[%]
40.0	--	--	--	--	--	--	--	--	--	--	--	0	--		--	--	--
16.0	--	--	--	--	--	--	--	--	--	--	--	0	--		--	--	--
6.40	--	--	96	--	85	--	96	102	125	96	600	6	100±	13	13	-2	no
2.56	114	108	88	88	--	83	--	--	83	--	564	6	94±	13	14	5	no
1.02	95	91	103	98	91	96	101	--	130	89	894	9	99±	12	13	-1	no
Control	102	91	97	101	94	83	123	85	82	127	985	10	99±	16	16	—	—

conc.      = concentration

N           = number of the surviving parental daphnids producing living juveniles

Red.       = reduction of the reproductive output (a negative value means increase of the reproductive output)

Stat.       = statistical significance(Kruskal-WallisOne Way Analysis of Variance on Ranks, P = 0.665)

no         = statistically not significant

--         = not applicable, due to the mortality of the parental daphnid

Table 7: Intrinsic Rates of Natural Increase

Nominal test item concentration [mg/L]	Intrinsic rate of natural increase in replicate no.										Mean IR		CV
	1	2	3	4	5	6	7	8	9	10	MV±SD		[%]
40.0	--	--	--	--	--	--	--	--	--	--	--		--
16.0	--	--	--	--	--	--	--	--	--	--	--		--
6.40	--	--	0.49	--	0.48	--	0.48	0.49	0.52	0.49	0.49±	0.01	3
2.56	0.49	0.47	0.46	0.47	--	0.48	--	--	0.43	--	0.47±	0.02	4
1.02	0.49	0.46	0.49	0.50	0.50	0.49	0.49	--	0.51	0.44	0.49±	0.02	4
Control	0.47	0.47	0.49	0.50	0.45	0.47	0.47	0.46	0.49	0.48	0.48±	0.01	3

--         = not applicable, due to the mortality of the parental daphnid(s)

Table 8: First Appearance of Living Juveniles and Mean Number of Broods in the Individual Groups

Nominal test item concentration [mg/L]	Day of first appearance of living juveniles										First	Mean
	in replicate no.										appearance	number
	1	2	3	4	5	6	7	8	9	10	mean day	of broods
40.0	--	--	--	--	--	--	--	--	--	--	--	--
16.0	--	--	--	--	--	--	--	--	--	--	--	--
6.40	--	--	8	--	8	--	8	8	8	8	8.0	4.0
2.56	8	8	8	8	--	8	--	--	9	--	8.2	4.2
1.02	8	8	8	8	8	8	8	--	8	9	8.1	4.1
Control	9	8	8	8	8	8	9	8	8	9	8.3	4.4

--         = not applicable, due to the mortality of the parental daphnid(s)

Table 9: Stillborn Juvenilesand Aborted Eggs after 21 Days

Nominal test item concentration   [mg/L]	Number of		Total no.		Mean stillborn juveniles and aborted eggs per parent alive MV±SD
	stillborn juveniles	aborted eggs
			å	N
40.0	--	--	--	0	--
16.0	--	--	--	0	--
6.40	0	4	4	6	0.667±1.03
2.56	0	1	1	6	0.167±0.408
1.02	0	0	0	9	0±0
Control	0	0	0	10	0±0

N  = number of the surviving parental daphnids

Table 10: Relative Number of Dead (Stillborn + Aborted Eggs)to Total Number of Juveniles

Nominal test item concentration [mg/L]	Number of juveniles			Percentage of dead juveniles^#) [%]
	Dead	Alive	Total
40.0	--	--	--	--
16.0	--	--	--	--
6.40	4	600	604	< 1
2.56	1	564	565	< 1
1.02	0	894	894	0
Control	0	985	985	0

Dead = aborted eggs + stillborn juveniles

Total = dead + alive juveniles

^#)       = related to the total number of juveniles

--      = not applicable, due to the mortality of the parental daphnid(s)

Validity criteria fulfilled:

yes

Conclusions:

The adult mortality was the most sensitive effect in this study. Based on the significant adult mortality at the concentration levels of
2.56 to 40.0 mg/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 1.02 mg/L and the
Lowest Observed Effect Concentration (LOEC) was assessed at 2.56 mg/L.
Based on the nominal test item concentrations, the EC10 was observed at 1.02 mg/L. The EC50 was calculated by sigmoidal dose-response
regression to be 5.26 mg/L (95% confidence limits: 2.23 – 11.8 mg/L).
Due to the lack of significant effects, no EC-values for the reproduction could be calculated.

Executive summary:

The Daphnia magna Reproduction Test (semi-static, 21 d) was conducted according to OECD 211 (2012). Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids, individually held, were used per concentration level, and control. The study was carried out under semi-static conditions with concentrations ranging from 1.02 to 40.0 mg/l. The measured test item concentrations were all within ± 20 % of the nominal concentrations throughout the exposure period indicating that the test item concentrations were successfully maintained for the duration of the test.

Aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects or parameters such as intrinsic rate of natural increase, number of broods with living juveniles, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality and body length of the parental daphnids.

The adult mortality was the most sensitive endpoint in this study. Based on the significant adult mortality at the concentration levels of 2.56 to 40.0 mg/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed as 1.02 mg/L and theLowest Observed Effect Concentration (LOEC)was assessed as 2.56 mg/L.
Based on the nominal test item concentrations, the EC₁₀was determined as 1.02 mg/L. The EC₅₀was calculated to be 5.26 mg/L (95% confidence limits: 2.23 – 11.8 mg/L). Due to the lack of significant effects, no EC-values for the reproduction could be calculated.

Description of key information

The Daphnia magna Reproduction Test (semi-static, 21 d) was conducted according to OECD 211 (2012). Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids, individually held, were used per concentration level, and control. The study was carried out under semi-static conditions with concentrations ranging from 1.02 to 40.0 mg/l. The measured test item concentrations were all within ± 20 % of the nominal concentrations throughout the exposure period indicating that the test item concentrations were successfully maintained for the duration of the test.
Aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects or parameters such as intrinsic rate of natural increase, number of broods with living juveniles, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality and body length of the parental daphnids.The adult mortality was the most sensitive endpoint in this study. Based on the significant adult mortality at the concentration levels of 2.56 to 40.0 mg/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed as 1.02 mg/L and theLowest Observed Effect Concentration (LOEC)was assessed as 2.56 mg/L. Based on the nominal test item concentrations, the EC10was determined as 1.02 mg/L. The EC50was calculated to be 5.26 mg/L (95% confidence limits: 2.23 – 11.8 mg/L). Due to the lack of significant effects, no EC-values for the reproduction could be calculated.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

1.02 mg/L