Amines, N-(C16-18 and C18-unsatd. alkyl)-N,N',N'-trimethyltrimethylenedi-

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:

2017-05-02 to 2017-05-24, with the definitive exposure phase from 2017-05-03 to 2017-05-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The test item concentrations of N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine were analytically verified via LC-MS/MS in fresh media at the start of the exposure intervals on days 0, 9, 14 (0 hours) and in old media at the end of the exposure intervals on days 2, 12, 16 (48 and 72 hours, respectively) in all concentration levels with surviving parental daphnids and the control. Details of the analytical method are presented.

Sampling for the analytical monitoring (aqueous phase)
At the start of the exposure intervals, samples of the fresh media
were taken after preparation of all test item concentrations and analyzed.
At the end of the exposure intervals (48 or 72 hours), samples of the old media were taken preferably from the test vessels.

Criteria for the analytical monitoring (target)
Recoveries of the test item should be within ± 20% of the initially
measured concentrations.

Determination of adsorption
Adsorption to glass walls of the test vessels was analytically verified
of the test item on glass once during the study in two concentration levels (e.g. 6.40 and 40.0 µg/L) at the end of the longest exposure interval (72 hours) on study day 12. After sampling for the test medium analysis (aqueous phase), the glassware was emptied and an appropriate amount of a suitable solvent was added. The concentration of the test item in this solution was measured and the adsorbed test item amount was calculated from this concentration. Before this procedure, the test vessels were carefully rinsed with demineralized water to remove the remaining algae and test media.

Vehicle:

no

Details on test solutions:

Definitive Test
The definitive reproduction test was carried out with five concentration levels of the test item N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine in the range of 2.56 to 100 µg/L, prepared with natural river water as specified in Table 5, which was supplemented with the mineral components of the culture medium as specified in Table 4. The definitive test was performed under semi-static conditions with a water renewal three times per week.

Preparation of the stock solution
A stock solution (1000 µg/L of the test item were weighed out) was freshly prepared with dilution water (see Table 4) for each exposure interval. The stock solution was mixed thoroughly by manual agitation and was used for preparation of the test concentrations.

Test concentrations
5 concentration levels of the test item in a geometric series with a separation factor of 2.5, prepared by diluting the stock solution of 1000 µg/L with dilution water, were tested as follows:
2.56 - 6.40 - 16.0 - 40.0 - 100 µg/L

The test concentrations were selected based on the results of a non-GLP preliminary range finding test. For results, see section 7.1.

Control
10 replicates of dilution water without test item

Test method
The study was performed under semi-static conditions with a renewal of the test solutions 3 times per week (i.e. on Monday, Wednesday and Friday).

Test duration
21 days

Test vessels
Glass beakers (5 (ID) x 8 (H) cm), 100 mL capacity, pre-conditioned with the test solutions and loosely covered with watch glasses

Coating of the test vessels
One day before the start of the exposure and each renewal of the test solutions, the glass beakers were pre-conditioned with the test solutions for 24 hours under test conditions in order to saturate the walls of the glassware. Thereafter, the test vessels were emptied and refilled with freshly prepared test solutions.

Test volume
50 mL

 
Test medium
Water of the river Grane was used as its properties in terms of organic matter content and suspended solids are considered typical for European rivers and are close to those in the EU risk assessment methods. The water parameters and the geographical site are given in Table 5.

The natural river water was supplemented with the mineral components for Elendt M4 medium (as specified in Table 4) to ensure a sufficient reproduction of the daphnids.

Table 5: Water Parameters of the River Water
River Grane
Location Im Granetal,38685 Langelsheim,Germany
Latitude: 51.9146°, Longitude: 10.3819°
Sampling Date 2016-12-20
Weather on Day of Sampling Cloudy, -1°C
Colour Yellowish to brownish, clear
pH 7.66
Conductivity [µS/cm] 152.4
Dissolved Oxygen [mg O2/L] 8.91
TOC [mg C/L] 2.71
Ammonium-N [mg NH4-N/L] < 0.04
Nitrate-N [mg NO3-N/L] 0.8
o-Phosphate-p [mg P/L] < 0.05
Total Phosphate [mg P/L] < 0.05
Suspended Matter [mg/L] 14.4
Total Hardness [mg CO3/L] 60.88
Storage conditions 15 ± 2°C

Storage conditions
The natural river water was stored at 15 ± 2 ° of the river water

Number of daphnids and replicates
10 daphnids in 10 replicates were used for all concentration levels and the control with one daphnid per replicate, which was held individually.

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 at the 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.

Acclimatization
The juvenile daphnids were acclimatized in the test medium (natural river water supplemented with mineral components of the culture medium as specified above) for at least 2 hours prior to insertion in the test solutions. An acclimatization of the brood daphnids 3 weeks prior to the test as suggested by the guideline was not necessary, since the test medium is supplemented with the same mineral components as the culture medium. 
Application 50 g test solution per replicate was weighed out into each test vessel. 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; Pseudokirchneriella subcapitata (0.789 - 1.14 mL) and Desmodesmus subspicatus (0.317 - 0.437 mL) suspension was provided as food corresponding to 0.2 mg C per Daphnia and day.
There was variation according to the density of the algae suspension, but it was the same for all test groups on each feeding day.

Test temperature (target)
The temperature was in the range of 18 – 22 °C and did not vary by more than 2°C.

Light intensity (target)
Max. 1500 lx

Photoperiod 1
6/8 hours light/dark cycle

Aeration
Test vessels were not aerated during the test.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system Daphnia magna STRAUS (Clone 5)

Reason for the selectionof the test system
Daphnia magna is the preferred species in accordance with the
test guideline and is bred at the test facility.
The 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 survived parent animal when cultured under the described conditions.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH,
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 hours illumination; light intensity of max. 20 µE.m-2 . s-1 (max. 1340 lx)

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L, is used. The composition of the culture medium is presented

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 > 10^6 cells/mL. The algae are cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany

Test type:

semi-static

Water media type:

other: natural river water

Limit test:

no

Total exposure duration:

21 d

Hardness:

Total Hardness [mg/L] as CaCO3
Nominal
test item concentration
[µg/L] fresh old fresh old fresh old
Day 0 Day 2 Day 09 Day 12 Day 14 Day 16
2017-05-03 2017-05-05 2017-05-12 2017-05-15 2017-05-17 2017-05-19
100 233 237 223 228 240 224
Control 240 231 235 226 233 231
The total hardness was above 140 mg/L as CaCO3.

Test temperature:

Temperature [°C]
Nominal
test item concentration
[µg/L] fresh old fresh old fresh old
Day 0 Day 2 Day 09 Day 12 Day 14 Day 16
2017-05-03 2017-05-05 2017-05-12 2017-05-15 2017-05-17 2017-05-19
100 20.7 20.6 20.4 20.5 20.7 20.6
Control 20.7 20.7 20.5 20.5 20.9 20.6
The temperature was within the range of 20 ± 1 °C.

pH:

pH-Values
Nominal
test item concentration
[µg/L] fresh old fresh old fresh old
Day 0 Day 2 Day 09 Day 12 Day 14 Day 16
2017-05-03 2017-05-05 2017-05-12 2017-05-15 2017-05-17 2017-05-19
100 7.99 7.76 8.90 8.03 8.04 7.48
Control 8.90 7.63 8.65 8.19 8.27 7.45
The pH-values should be in the range of 6 – 9. Significant deviations ≥ 1.5 units between the initial and final pH-values were not observed.

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
test item concentration
[µg/L] fresh old fresh old fresh old
Day 0 Day 2 Day 09 Day 12 Day 14 Day 16
2017-05-03 2017-05-05 2017-05-12 2017-05-15 2017-05-17 2017-05-19
100 8.84 9.21 8.36 8.19 8.44 7.97
Control 9.15 8.96 8.80 8.91 8.52 8.02
The dissolved oxygen concentration was above 3 mg/L.

Details on test conditions:

see detail on test solutions

Reference substance (positive control):

yes

Duration:

21 d

Dose descriptor:

LOEC

Remarks:

reproduction/ adult mortality

Effect conc.:

> 100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Remarks:

reproduction/ adult mortality

Effect conc.:

>= 100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

other: EC10/EC50

Effect conc.:

> 100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

other: LC20/LC50/LC100

Effect conc.:

> 100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Results with reference substance (positive control):

A reference test was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility. The results of the most recent test are presented in section 7.2.4.

Reference item Potassium dichromate p.a. (SIGMA)

Purity 99.0%

Batch No. MKBV0900V

Expiry date 2021-11-25

Test concentrations 1.00 - 2.00 - 4.00 mg/L

Ranges of validity EC50 (24 hours): 0.6 - 2.4 mg/L, according to AQS P 9/2 (clone 5)
EC50 (24 hours): 0.6 - 2.1 mg/L, according to OECD 202 (clone A)

Test duration 24 hours

Test vessels Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Volume of test medium 20 mL

 
Dilution water ISO test water, according to OECD 202, Annex 3

Loading of test organisms 20 animals, divided into 4 replicates, each with 5 animals

Test duration 2017-05-04 to 2017-05-05

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2017 05-04 to 2017-05-05. For results of the most recent of the monthly performed reference tests, see Table 22.

Table 22: EC50-Value (with 95% confidence limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 1.60 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
95% confidence limits 1.43 - 1.79 mg/L

Reported statistics and error estimates:

Determination of the The EC10 / 50-values for the reproductive output and their 95%
EC-/LC-values confidence limits were not calculated since no statistical significant reduction in reproductive output was observed.
The LC20 / 50-values for the adult mortality and their 95% confidence limits were calculated by probit analysis with the software ToxRat Professional. The LC100 was empirically derived from the observed data.
The EC50-value for the reference item was calculated by sigmoidal dose-response regression. The respective 95% confidence limits for the EC50-value of the reference item were calculated from the standard error and the t-distribution. These calculations were carried out from the best-fit values with the software GraphPad Prism.
 
Calculation of the The intrinsic rate of natural increase (IR) was calculated for the
intrinsic rate survived parental daphnids with the following EULER-LOTKA-equation with the ToxRat Professional.
 (e -A  IR) * Sx * Jx = 1
A = Age
IR = Intrinsic rate of natural increase
Sx = Survival rate on day x
Jx = Reproduction rate on day x

The intrinsic rate of natural increase is a measure of population growth which integrates reproductive output and age-specific mortality. In steady state populations, it will be zero. For growing populations, it will be positive and for shrinking populations, it will be negative. Clearly, the latter is not sustainable and ultimately will lead to extinction.

Software All data are computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- Excel, MICROSOFT CORPORATION
- GraphPad Prism, GraphPad Software, Inc.
- SigmaPlot, Systat Software, Inc.
- ToxRat Professional, TOXRAT SOLUTIONS GMBH

 

1.1.1.1  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 Table23 to Table28.

The average number of living juveniles at the end of the test after 21 days per survived parental daphnid was 93 in the control group and thus the validity criterion for the reproduction of a mean of ≥ 60 living offspring per survived parent animal in the control was met.

No parental daphnids were excluded due to accidental or inadvertent mortality from the evaluation of the reproductive output. No statistically significant reduction of the reproductive output in comparison to the reproductive output in the control was determined at all tested concentration levels 2.56 to 100 µg/L (Multiple sequentially-rejective Welsh t-test after Bonferroni-Holm,α = 0.05).

Therefore the EC₁₀ and EC₅₀ for the test item N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine was > 100 µg/L for both parameters. The NOEC and LOEC were≥ 100 and > 100 µg/L. For details, seeTable 1, Table 2 and Figure 1 to Figure3.

 

Figure1:    Mean Number±Standard Deviation of Living Juvenilesper Introduced Parental Daphnid

 

 

 

 

Figure2:     Concentration-Effect Curve showing the Influence on cumulative Offspring per Introduced Parental Daphnid as observed after 21 days

                     (based on the nominal concentrations of the test item [µg/L])

 

 

The parameter living offspring per survived parental animal at the end of exposure was also evaluated and the results were comparable to the cumulative offspring per introduced parent animal. Nevertheless, the evaluation based on the cumulative offspring per introduced parent is the ecologically most relevant response variable.

 

 

Figure3:    Mean Number±Standard Deviation of Living Juvenilesof the Parental Daphnids survived until the End of the Testafter 21 Days

 

1.1.1.2  Adult Mortality

 

Accidental and inadvertent mortality as defined in the OECD test guideline were not observed. Adult mortality was not observed in the control and the nominal test concentrations of 2.56 to 40.0 µg/L. Only in the highest test concentration of 100 µg/L a mortality of 10 % of the introduced adults was observed. No statistical significant difference was found for this parameter when tested with Fisher’s exact binomial test with Bonferroni correction (α = 0.05). Therefore, the LC₂₀- and LC₅₀-values were determined to be > 100 µg/L (nominal test item concentration).The LOEC was > 100 µg/L and the NOEC was ≥ 100 µg/L. For details, see Table 1, Table 2, Table 10 and Figure 4.

The adult mortality in the definitive test was lower than in the preliminary test. In the latter one the measured initial concentrations were close to the nominal concentrations whereas in the definitive test the results of two sampling dates were in the range of 61 to 81% of the nominal values. Moreover, the measured concentrations from 72 hours old media were lower. The presence of algae as food might have an influence as well.

Table10:    Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure

                (n = 10)

Nominal test item concentrations [µg/L]		Adult Mortality [%]
		7 days		14 days		21 days
100		10		10		10
40.0		0		0		0
16.0		0		0		0
6.40		0		0		0
2.56		0		0		0
Control		0		0		0

 

 

1.1.1.3  Intrinsic Rates of Natural Increase (IR)

 

Theintrinsic rates of natural increase (IR)of the survived parental daphnids accounting for generation time and number of offspring were used for calculation of population growth and maintenance. For details, see Table 3 and Table 11.

 

Table11: Intrinsic Rates of Natural Increase

Nominal test item concentrations [µg/L]		Intrinsic rate of natural increase in replicate no.											Mean IR			CV
		1		2		3		4		5		6		7		8		9		10		MV±SD			[%]
100		0.416		0.407		0.386		0.382		0.367		--		0.281		0.377		0.388		0.389		0.377 ±		0.0387		10.3
40.0		0.348		0.345		0.399		0.4		0.387		0.368		0.424		0.337		0.384		0.412		0.380 ±		0.0298		7.8
16.0		0.377		0.356		0.349		0.369		0.401		0.386		0.372		0.336		0.36		0.369		0.367 ±		0.0188		5.1
6.40		0.342		0.401		0.337		0.408		0.41		0.378		0.37		0.414		0.355		0.367		0.378 ±		0.0289		7.6
2.56		0.393		0.368		0.382		0.369		0.378		0.372		0.352		0.384		0.369		0.379		0.375 ±		0.0111		3.0
Control		0.432		0.386		0.395		0.382		0.356		0.354		0.362		0.385		0.331		0.387		0.377 ±		0.0278		7.4

 

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

 

1.1.1.4  First Appearance of living Juveniles

 

The first appearance ofliving juveniles was observed between days 8 and 9 at all introduced parental daphnids of the control and the concentration levels 2.56 to 100 µg/L. For details, see Table 3 and Table 12.

 

Table12: First Appearance of Living Juveniles in the Individual Groups

Nominal test item concentrations [µg/L]		Day of first appearance of living juveniles at the introduced parental daphnids in replicate no.											First
												appearance
		1		2		3		4		5		6		7		8		9		10		mean day
100		8		8		8		8		8		--		8		8		8		8		8.0
40.0		8		8		8		8		8		8		8		9		8		8		8.1
16.0		8		8		8		9		8		8		8		8		8		8		8.1
6.40		9		8		8		8		8		8		8		8		8		8		8.1
2.56		8		8		8		8		9		8		9		8		9		8		8.3
Control		8		8		8		8		8		8		9		8		9		8		8.2

*    = only broods of the survived parental daphnids with living juveniles taken into account

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

 

1.1.1.5  Stillborn Juveniles and Aborted Eggs

 

No stillborn juveniles or aborted eggs were observed in the control and in the concentration levels of 2.56, 6.40, 40.0 and 100 µg/L during the exposure period of 21 days (see Table 13 and Table14). In the concentration level of 16.0 µg/L four aborted eggs were observed on study day 11. Related to the total number of produced juveniles (dead + alive) per survived daphnid, the percentage of dead juveniles was ≤60% in the control and all concentration levels (Table 3).

 

Table13:    Stillborn Juvenilesand Aborted Eggs produced by the Survived Parental Daphnids after 21 Days

Nominal test item concentrations [µg/L]		Number of			Total no.   å		Number of survived adults N
		stillborn juveniles		aborted eggs
100		0		0		0		9
40.0		0		0		0		10
16.0		0		4		4		10
6.40		0		0		0		10
2.56		0		0		0		10
Control		0		0		0		10

N   = number of the survived parental daphnids

Table14:    Relative Number of DeadJuveniles and Aborted Eggs to Total Number of Juveniles per Survived Parental Daphnids after 21 Days

Nominal test item concentrations [µg/L]		Number of				Percentage of dead juveniles^#) [%]
		Dead		Alive			Total
100		0		898		898		0
40.0		0		938		938		0
16.0		4		918		922		0
6.40		0		1008		1008		0
2.56		0		932		932		0
Control		0		931		931		0

Dead   = aborted eggs + stillborn juveniles

Total  = dead + alive juveniles

^#)          = related to the total number of juveniles

 

 

1.1.1.6  Growth (Total Body Length and Mean Dry Weight) of the Survived Parental Daphnids

 

The mean values of the body length (excluding the anal spine) of the survived parental daphnids in the tested concentration levels 2.56 to 100 µg/L were in the range of 4.47 to 4.83 mm per daphnid and 4.73 mm per daphnid in the control group.

The mean dry weight of the survived parental daphnids was in the range of 0.93 to 1.26 mg per daphnid in the concentration levels 2.56 to 100 µg/L and 1.28 mg per daphnid in the control (Table 3 and Table 15).

 

Table15:    Total Body Length and Dry Weight of the Survived Parental Daphnids

Nominal test item concentrations [µg/L]	Total length of the survived parent animals [mm]										MV	N	Dry weight [mg]
	Replicate no.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
100	4.50	4.75	4.50	4.50	4.75	--	4.50	4.25	4.50	4.00	4.47	9	8.4	0.93
40.0	4.50	4.50	4.75	4.75	4.75	5.00	5.00	4.50	4.50	4.50	4.68	10	12.5	1.25
16.0	4.75	4.50	4.75	4.75	5.00	5.00	4.25	4.75	4.75	4.75	4.73	10	12.6	1.26
6.40	4.75	5.00	5.00	4.75	5.00	4.75	4.75	4.75	5.00	4.50	4.83	10	11.2	1.12
2.56	5.00	4.75	5.00	4.25	4.75	4.50	4.00	4.50	4.75	4.75	4.63	10	12.3	1.23
Control	5.00	4.50	4.75	5.00	4.75	4.75	4.50	4.50	4.75	4.75	4.73	10	12.8	1.28

N  = number of the survived parental daphnids

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

 

 

 

1.1.1.7  Presence of Males

 

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

 

 

1.1.1.8  Occurrence of Ephippia (Winter Eggs)

 

No ephippia were observed in the control or in the test groups during the test.

 

 

Validity criteria fulfilled:

yes

Conclusions:

The effects on reproduction were evaluated based on the reproduction per introduced parent animal, since this parameter is the ecologically most relevant response variable and is required by the OECD test guideline 211 (2012), when a significant trend in mortality is detected.
A summary of the assessed effect levels based on the nominal concentrations of the test item N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine is given in the table below.
The overall effect threshold for effects of the test item under the test conditions was ≥ 100 µg/L (NOEC) and > 100 µg/L (LOEC) based on the nominal concentrations of the test item N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine.

All effect levels are based on nominal test item concentrations considering the performed bulk approach. There was no significant sorption to glass walls of the test vessels. Therefore, the nominal test item concentrations were used for the evaluation.
 
Copy of Table 1: Endpoints for Reproduction and Mortality
(based on the nominal concentrations of the test item)
Effect values N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine
Nominal test item concentrations
[µg/L]
EC10 Reproduction > 100
EC50 Reproduction > 100
LOEC Reproduction > 100
NOEC Reproduction ≥ 100
LC20 Adult mortality after 21 days > 100
LC50 Adult mortality after 21 days > 100
LC100 Adult mortality after 21 days > 100
LOEC Adult mortality after 21 days > 100
NOEC Adult mortality after 21 days ≥ 100

Executive summary:

A Daphnia magna reproduction test (semi-static, 21 d) of the test item N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine (batch number: 211401A Morris) was conducted at the test facility according to OECD 211 (2012) from 2017 -05 -02 to 2017 -05 -24, with the definitive exposure phase from 2017-05-03 to 2017-05-24.

Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids, held individually, were used per concentration level and control. At test start the daphnids were 2 to 24 hours old. The study was carried out undersemi-static conditions in natural river water with a medium renewal three times per week (i.e. on Monday, Wednesday and Friday). Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids.

 

The test item N-C16 -18 -alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3 -diamine is an amber liquid UVCB substance, which is dispersible in water. Nominal concentrations of the test item N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine were selected based on the results of an acute immobilization test as follows: 2.56 -6.40 -16.0 -40.0 -100 µg/L(separation factor of 2.5), prepared with natural river water as specified in Table5, which was supplemented with the mineral components of the culture medium as specified in Table4.

 

A stock solution with a nominal concentration of 1000 µg/L was prepared with natural river water which was supplemented with the mineral components of the culture medium as specified in section 4.2 and was used for the preparation of five concentration levels in the range of 2.56 to 100 µg/L.

 

The concentrations of three components of the test item N-C16 -18 -alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3 -diamine were analytically verified via LC-MS/MS in fresh media at the start of the exposure intervals on days 0, 9, 14 (0 hours) and in old media at the end of the exposure intervals on days 2, 12, 16 (48 and 72 hours, respectively) in all concentration levels with surviving parental daphnids and the control. Details of the analytical method are presented in section 14.

 

The measured concentrations of three componentsof the test item in fresh media at the start of the respective exposure intervals (0 hours) were in the range of 61 to 114% of the nominal concentrations for all components. At the end of the respective exposure intervals (48 and 72 hours), the measured concentrations of three components of test item in the old media were in the range of < LOQ to 44% of the nominal concentrations.The analytical results are presented in Table 16 and Table 17.

 

Adsorption to glass walls of the test vessels was analytically verified once during the study in two concentration levels (6.40 and 40.0 µg/L) at the end of one exposure interval (72 hours). Adsorption to the glass walls was negligible. For details, see section 7.2.2.

 

The environmental conditions were within the acceptable limits.The validity criteria of the test guideline were met (for details see section 8).

 

 

The aquatic ecotoxicity tests with N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diaminewere performed in river water to allow a PEC_aquatic,bulk/PNEC_aquatic,bulk approach and is considered to be conservative but more environmentally realistic than the standard method. This approach is based on PEC estimations representing 'total aquatic concentrations'. To characterize the risk to the aquatic compartment the PEC_aquatic,bulk is compared with the PNEC_aquatic,bulk derived from river water ecotoxicity studies (ECETOC, 2003).

 

In order to class standard laboratory toxicity study valid, it is of particular importance that – besides information on test substance, test method / conditions and test organism used – suitable precautions are taken to prevent the loss of test substance by adsorption and that exposure concentrations are based upon measured levels.

 

For ecotoxicity tests performed using the bulk approach, however, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware should be accounted for. For a valid bulk approach test the concentration-effect relationship should be based on the sum of adsorbed and dissolved substance in the volume of the medium tested. One of the advantages of the bulk approach tests with these difficult substances is that in the presence of suspended matter, humic acids and/or algae, the residual sorption to glassware will be negligible. The results of these bulk approach tests are therefore much easier and more realistic, and if compared to PEC_bulk clearly provide a more appropriate assessment of risks for the environment. An individual test design was applied. Natural river water was used as dilution medium (for details please refer to page 17).

 

The effects on reproduction were evaluated based on the reproduction per introduced parent animal. A summary of all endpoints based on the nominal concentrations of the test item N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine is given in Table1.

 

Table1:      Endpoints for Reproduction and Mortality

(based on the nominalconcentrations of the test item)

Effect values	N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine Nominal test item concentrations [µg/L]
EC10 Reproduction	> 100
EC50 Reproduction	> 100
LOECReproduction	> 100
NOECReproduction	≥100
LC20 Adult mortality after 21 days	> 100
LC50 Adult mortality after 21 days	> 100
LC100 Adult mortality after 21 days	> 100
LOECAdult mortality after 21 days	> 100
NOECAdult mortality after 21 days	≥100

 

Description of key information

One long-term toxicity test with aquatic invertebrates is available for N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine. The EC10 observed in this study is >100 µg a.i./L  The observed long term (21d) EC50 for adult mortality is >100 µg a.i./L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.1 mg/L

Additional information

A Daphnia magna reproduction test (semi-static, 21 d) with N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine was conducted according to OECD 211 (2012).

Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids, held individually, were used per concentration level and control. At test start the daphnids were 2 to 24 hours old. The study was carried out undersemi-static conditions in natural river water with a medium renewal three times per week (i.e. on Monday, Wednesday and Friday). Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids.

 

N-C16 -18 -alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3 -diamine is an amber liquid UVCB substance, which is dispersible in water. Nominal concentrations of N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine were selected based on the results of an acute immobilization test as follows: 2.56 -6.40 -16.0 -40.0 -100 µg/L(separation factor of 2.5), prepared with natural river water, which was supplemented with the mineral components of the culture medium.

 

The concentrations of the three main components of N-C16 -18 -alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3 -diamine were analytically verified via LC-MS/MS in fresh media at the start of the exposure intervals on days 0, 9, 14 (0 hours) and in old media at the end of the exposure intervals on days 2, 12, 16 (48 and 72 hours, respectively) in all concentration levels with surviving parental daphnids and the control.

 

The measured concentrations in fresh media at the start of the respective exposure intervals (0 hours) were in the range of 61 to 114% of the nominal concentrations for all components. At the end of the respective exposure intervals (48 and 72 hours), the measured concentrations were in the range of < LOQ to 44% of the nominal concentrations.

 

Adsorption to glass walls of the test vessels was analytically verified once during the study in two concentration levels (6.40 and 40.0 µg/L) at the end of one exposure interval (72 hours). Adsorption to the glass walls was observed to be negligible. 

 

The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met.

  

The aquatic ecotoxicity tests with N-C16-18-alkyl-(evennumbered, C18 unsaturated) trimethylpropane-1,3-diamine were performed in river water to allow a PECaquatic,bulk/ PNECaquatic,bulk approach (ECETOC 2003) and is considered to be conservative but more environmentally realistic than the standard method.

The results of these bulk approach tests are much easier to interpret and more realistic, and when compared to PECbulk clearly provide a more appropriate assessment of risks for the environment.

Following the bulk approach and considering that biodegradation as possible reason for the observed decrease is very unlikely considering the short time frame of refreshment and the fact that the observed sorption to glassware is negligible shows that the test organisms were fully exposed to the test substance during the test. It is therefore considered justified to use the nominal test concentrations to derive the effect values.

 

The effects on reproduction were evaluated based on the reproduction per introduced parent animal.

Under the study conditions, the 21d EC10 and EC50 for reproduction are >100 µg/L. The LC50 for adult mortality is > 100 µg/L.