Amines, N'-[3-[(3-aminopropyl)amino]propyl]-N,N-di-C16-18-alkyltrimethylenedi-

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:

From 08-09-2015 till 06-10-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP, all validity criteria fulfilled, Identity data complete, including chemical analyses.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples of all test concentrations were taken in all old and new solutions.
10 mL was sampled for fresh solutions and a larger 25 mL sample was initially sampled for old solutions to aid extraction. Standard leaching solution (see analytical report) was added to new samples in a 1:1 ratio prior to analysis. Acidified leaching solution (see analytical report) was used for the old samples as this improved the recoveries slightly. Sampling in the old solutions was altered by amendment in an effort to improve the recovery in the old samples. During these samples 20 mL samples were taken in a 1:1.5 ratio with acidified leaching solution.

Vehicle:

no

Details on test solutions:

Test solutions
Preparation of the stock solutions
The test substance is dispersible in water. To prepare the stock solutions for every water renewal, on between 0.0014 and 0.0048 g of test substance was weighed on an analytical balance to which 100 or 200 mL of test medium was added. The stock solutions were agitated mechanically for at least 45 seconds until the test material was completely dispersed and no visible particles remained. Stock solution concentrations varied between 14 and 24 mg/L. The pH of the stock solution was checked and found to be between 7.4 and 8.2 and was not adjusted further. A fresh stock solution was prepared for each solution change in the same manner. The first stock was made and discarded to coat the glassware and minimize the initial loss due to adsorption to glass.

Preparation of the test solutions
Test solutions were prepared by further dilution of the relevant stock solutions with test medium in volumetric flasks. All pipetting took place while stock was under agitation and all pipettes were rinsed at least once and the contents discarded if being used for the first time. The ratio between two consecutive test concentrations was 2.2. Test vessels were filled directly from volumetric flasks immediately after preparation. The solutions were renewed three times a week during the test.

The pH of the test solutions should be between 6 and 9 at refreshment and close to the value of the test medium.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test animals
The test animals were taken from a Daphnia magna clone 5 stock, (Origin: WIL Research Europe, The Netherlands) cultured in conformity with the relevant SOP. The parent animals were cultured in test medium from the day they were born.
The animals used in the test were less than 24 hours old and were obtained from parent animals reproducing parthenogenically and having an age of 2-4 weeks (having previously produced at least one brood before use).The culture is checked half-yearly for sensitivity by a reference test with potassium dichromate. The most recent reference test results were within the range given in the guideline and the culture was deemed suitable for use.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

Water hardness was 13.6 °dH which is equal to 242 mg/L as CaCO3

Test temperature:

Temperature range observed in fresh and used solutions monitored at the moment of renewal: min. 20.5; max. 21.4 °C.
Temperature in the test room checked with a digital thermometer ranged from: 19.4 to 21.5 °C which fell outside of the ±1ºC as indicated in the study plan.

pH:

The pH range observed in fresh and used solutions monitored at the moment of renewal: min. 6.9; max.8.3.

Dissolved oxygen:

The observed oxygen concentration range: min. 7.3; max. 10.7 mg O2/L.

Nominal and measured concentrations:

Test concentrations
The following nominal test concentrations were used: 0.02, 0.044, 0.097, 0.213 and 0.47 mg/L.
Which correspond with the following Time Weighted Mean concentrations: 0.01, 0.021, 0.046, 0.103, 0.204 mg/L

Details on test conditions:

Test vessels
50 mL (nominal) glass beakers were used, containing approximately 50 mL of test solution and covered by glass plates during the test.

Test room, temperature control and light regime
The test was carried out in a temperature-controlled room. The light regime was 16 h of ambient light per day, provided by fluorescent tubes.

Test medium
Reconstituted water (M4) according to Elendt (Elendt, 1990) was used for culturing and in the test. The pH of this solution is typically 8.0 ± 0.5 and water hardness is typically between 140 and 250 mg/L (as CaCO3). The air was water-saturated and purified by activated charcoal and a cotton filter.

Test procedure
At the start of the test the daphnids were randomly distributed to the test vessels. For each test concentration and control, ten vessels, with one daphnid per vessel were tested. Parent animals were checked for immobilisation on at least 6 days per week of the test, by gently shaking the test vessel. From the day of the first brood, observations of broods (aborted, living and dead progeny) were also made in each container at each concentration. The day of brood release and the number of living and dead neonates per brood or abortions were noted. Any other observations of abnormalities were also recorded.

At the end of the test, the length of all surviving parent animals was measured to the nearest 0.1 unit using a binocular microscope (one unit was equal to = 1.5 mm). Following measurement of body length (including spine), parent animals were placed in an oven at 105 °C overnight in groups and then weighed to allow calculation of the mean individual weight per surviving parent animal per concentration. The test duration was 21 days.

Reference substance (positive control):

yes

Remarks:

Twice a year

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

148 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

236 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

204 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

Parental

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

103 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

other: Adult length

Details on results:

The following quality criteria were respected:

• Cultures were in good health (i.e. disease free, no ephippia or males, no discolored animals, valid reference test).
• Two parent animals died in the control group over the test period, which is not more than 20%.
• The average number of juveniles per parent animal alive at the end of the test in the control was 138 after 21 days (minimum acceptable = 60)
• Analytical quality criteria were met see Table II
• The validity criterion for the coefficient of variation (less than 25% in the control based on the number of living neonates for each parent animal alive at the end of the test) was achieved.

Results with reference substance (positive control):

Within the guideline requirements

Reported statistics and error estimates:

Probit analysis for the derivation of the ECn values
Williams multiple sequential t-test for the derivation of the NOEC.

Ia. Measured concentrations in µg/L of fresh, used and stock solutions quantified using the C16:18 component.

Conc.	D 0 (F)	D2 (O)	D2 (F)	D4 (O)	D4 (F)	D7 (O)	D7 (F)	D9 (O)	D9  (F)	D 11 (O)	D11 (F)	D 14  (O)	D14  (F)	D 16 (O)	D 16 (F)	D 8 (O)	D 18 (F)		D 21 (O)
C	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ	<LOQ		<LOQ	<LOQ
20	9.4	3.5	17.8	5.7	17	9.1	10	13.8	16.4	94.9	18.1	18.6	10.3	10	15.2	12.6	12.9		2.5
44	28	6.4	64	10	32.6	12.2	17.5	16.4	31.3	16	38.7	11.2	26.1	14.5	29.5	23.7	33.1		8.2
97	76.9	13.5	87.4	17.2	84	24.5	28.5	28.7	70.3	46.1	93.7	107.5	56.6	34.2	77.9	44.5	81.4		24.4
213	215.6	25.3	185.7	72.6	174.5	44.4	54.8	46.6	159.2	67.9	102	60	123.3	64.3	146	87.3	152		74.9
470	392.2	64.5	441.7	68.1	507.5	57.4	255.1	106.2	420.9	187.9	564.5	132.9	389.7	117	225.3	190	368.8		96.6
Stock	12476		13257		16880		10519		15776		22354		14810		17646		19867

Figures in red indicate results that were extracted from the old samples with a more severe extraction method. This method was considered poorly reproducible and the figures were excluded from the time weighted average calculations. The accompanying new measurements (shaded grey) were therefore also excluded. F=Fresh, O=Old, D= Day.

 

Table Ib. Time weighted mean (TWM) concentrations of test samples in µg/L quantified using the C16:C18 component.

Calculated as indicated the OECD 211 guideline. Used for statistical evaluations of the endpoints.

 

Nominal Concentration	TWM
C	<LOQ
20	10
44	21
97	46
213	103
470	204

 

Table III. Number of living juveniles per parent animal at each nominal test concentration, mean number of neonates per concentration and coefficient of variance.

1	144	132	141	158	120	M
2	159	M	166	M	M	75
3	143	115	152	124	157	M
4	134	144	M	136	136	105
5	128	M	141	126	120	M
6	M	94	M	113	116	114
7	146	143	147	M	M	99
8	135	126	133	M	136	60
9	M	138	138	132	152	107
10	114	148	171	171	153	71
Total	1103	1040	1189	960	1090	631
Mean	138	130	149	137	136	90
Stdev	13	18	14	20	16	21
CV%	10	14	9	15	12	23

 

M: parent animal died during the test

St. dev.: Standard deviation

CV: Coefficient of variation

 

 

Validity criteria fulfilled:

yes

Conclusions:

The EC10 and EC50 for reproduction, were calculated as 0.148 and 0.236 mg/L (148 μg/L and 236μg/L) respectively. The NOEC for reproduction was determined as 0.103 mg/L (103 μg/L). The NOEC for adult mortality was determined as 0.204 mg/L (204 μg/L). The NOEC for adult length was determined as 0.103 mg/L (103 μg/L).

Executive summary:

The purpose of this study was to assess the toxicity of the test substance dissolved in fresh water, on the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a semi-static test complying with the OECD Guideline No. 211 (OECD, 2012).

The primary test criterion used to indicate the toxicity of the test substance was reproductive capacity expressed as the total number of neonates per surviving parent animal at the end of the study due to their being no test substance related parent mortality. Data on parental length and dry-weight was also generated for use in determining secondary endpoints as required.

The nominal concentrations used in the study were as follows: 0.02, 0.044, 0.097, 0.213 and 0.47 mg/L.

All concentrations given refer to the technical product as supplied by the sponsor. Analytical determinations of the test solutions were made on all fresh and old solutions as well all stock solutions throughout the test. The test substance concentration was found not to be stable according to the guideline criteria. Time weighted mean measured concentrations were therefore calculated, on which the endpoints were based. Quantification was carried out using the C16:C18 component with a precursor ion mass of 665.7. This was considered most representative of the test substance.

The following quality criteria were respected:

· Cultures were in good health (i.e. disease free, no ephippia or males, no discolored animals, valid reference test).

· Two parent animals died in the control group over the test period, which is not more than 20%.

· The average number of juveniles per parent animal alive at the end of the test in the control was 138 after 21 days (minimum acceptable = 60)

· Analytical quality criteria were met.

· The validity criterion for the coefficient of variation (less than 25% in the control based on the number of living neonates for each parent animal alive at the end of the test) was achieved.

The EC10 and EC50 for reproduction, were calculated as 0.148 and 0.236 mg/L (148 μg/L and 236μg/L)

respectively. The NOEC for reproduction was determined as 0.103 mg/L (103 μg/L). The NOEC for adult mortality was determined as 0.204 mg/L (204 μg/L). The NOEC for adult length was determined as 0.103 mg/L (103 μg/L).

Description of key information

One long-term toxicity test with aquatic invertebrates is available for tetramine di-C16-18. The EC10 observed in this study is 0.148 mg a.i./L based on based on time weighted mean measured concentrations.  The observed long term (21d) EC50 for reproduction was 0.236 mg a.i./L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.148 mg/L

Additional information

One long-term toxicity to aquatic invertebrates test is available for tetramine di-C16 -18. The purpose of this study was to assess the effect of tetramine di-C16 -18 dissolved in fresh water, on the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a semi-static test complying with the OECD Guideline No. 211 (OECD, 2012).

The primary test criterion used to indicate the toxicity of the test substance was reproductive capacity expressed as the total number of neonates per surviving parent animal at the end of the study due to their being no test substance related parent mortality. Data on parental length and dry-weight was also generated for use in determining secondary endpoints as required.

The nominal concentrations used in the study were: 0.02, 0.044, 0.097, 0.213 and 0.47 mg/L.

All concentrations given refer to active ingredient as the test substance is a UVCB. Analytical determinations of the test solutions were made on all fresh and old solutions as well all stock solutions throughout the test. Quantification was carried out using the C16:C18 component with a precursor ion mass of 665.7 Dalton. This was considered most representative of the test substance.

The test substance concentration was found not to be stable according to the guideline criteria. Time weighted mean measured concentrations were therefore calculated, on which the endpoints were based.

The EC10 and EC50 for reproduction, were calculated as 0.148 and 0.236 mg/L (148 μg/L and 236μg/L)

respectively. The NOEC for reproduction was determined as 0.103 mg/L (103 μg/L). The NOEC for adult mortality was determined as 0.204 mg/L (204 μg/L). The NOEC for adult length was determined as 0.103 mg/L (103 μg/L).