Amines, di-C16-18-alkyl

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a long-term aquatic toxicity study on invertebrates is available

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-03-24 to 2010-04-14

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:

- Concentrations: The limit concentration and the control were analysed at least once within 7 days in the fresh (0 h) and old media (48 or 72 h).
- Sampling method: For the longest exposure interval of 72 h samples were taken from the fresh (0 h) and old media (72 h) once within the test period, respectively.
For the analyses of the old media separate replicates without algae and test organisms will be prepared and stored under test conditions.
Additional sampling may be carried out with respect to the findings. The sorption of the test item to the glassware was quantified once during the test period in a glass beaker, which has not been exposed before. Therefore, one test vessel of the limit concentration, containing daphnids and algae, was emptied after 72 hours of exposure and rinsed twice with demineralised water. Thereafter an extraction of the test vessel was carried out.
- Sample storage conditions before analysis: All samples were stored at room temperature until analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The water soluble fraction (WSF) with a nominal loading of 10 mg/L was prepared with dilution water two days prior to
application. The WSF was shaken with 20 rpm for 24 h at room temperature. After a separation phase of at least 24 h the WSF was taken from the homogeneous liquid phase. There was no filtration applied to prepare the WSF.
- Eluate: Natural river water
- Differential loading: The water soluble fraction (WSF) with a nominal loading of 10 mg test item/L was tested as limit concentration corresponding to geometric mean measured concentration of 0.0483 mg/L.
- Controls: 10 replicates of dilution water without test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna STRAUS is recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 days
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: 0.2 mg C/daphnia per day
- Frequency: daily


ACCLIMATION
- Acclimation period: 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: During acclimation the daphnids were not fed
- Feeding frequency: None
- Health during acclimation (any mortality observed): Healthy

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The parent animals were removed from the culture medium and the juveniles collected over a sieve and flushed into fresh medium

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Hardness:

Total Hardness [mg/L] as CaCO3
Geometric
Mean Measured
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 5 Day 7 Day 14 Day 16
Mar. 24th Mar. 26th Mar. 29th Mar. 31st Apr. 7th Apr. 09th
0.0483 79 81 90 87 97 89
Control 81 80 90 99 91 75

Test temperature:

Temperature [°C]
Geometric
Mean Measured
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 5 Day 7 Day 14 Day 16
Mar. 24th Mar. 26th Mar. 29th Mar. 31st Apr. 7th Apr. 09th
0.0483 21.6 21.5 21.0 21.2 21.3 20.8
Control 19.1 21.6 19.1 20.6 20.1 20.8

pH:

pH-Values
Geometric
Mean Measured
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 5 Day 7 Day 14 Day 16
Mar. 24th Mar. 26th Mar. 29th Mar. 31st Apr. 7th Apr. 09th
0.0483 7.64 7.80 7.84 7.47 7.83 7.35
Control 7.60 7.87 7.67 7.61 7.75 7.39

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Geometric
Mean Measured
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 5 Day 7 Day 14 Day 16
Mar. 24th Mar. 26th Mar. 29th Mar. 31st Apr. 7th Apr. 09th
0.0483 8.57 9.16 8.59 8.08 8.86 7.01
Control 9.38 9.10 9.20 8.67 9.57 7.41

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to information in materials and methods incl. tables

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: 100 mL glass beaker, filled with 50 mL test solution
- Aeration: No
- Renewal rate of test solution (frequency): 3 x per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural river water of the river Leine was used. This river is located near D-31171 Nordstemmen, Marienbergstraße, Germany (52° 10’ 14.98‘’, 9° 46’ 7.64’’).
River: Leine
Location D-31171 Nordstemmen
Sampling Date 2009-12-15
Weather on Day of Sampling cloudy, ca. -1 °C
Colour Yellowish, clear
pH 7.97
Conductivity [µS/cm] 386
Dissolved Oxygen [mg O2/L] 8.62
DOC [mg C/L] 3.9
TOC [mg C/L] 3.9
Ammonium-N [mg N/L] 0.042
Nitrate-N [mg N/L] 2.62
Total Nitrogen [mg N/L] 3.53
o-Phosphate-p [mg P/L] 0.062
Total Phosphate [mg P/L] 0.053
Suspended Matter [mg/L] 16.2
Total Hardness [mg CO3/L] 154
Storage conditions < 10 °C *


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark per day
- Light intensity: Max. 20 µE*m-2*s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Adult mortality: daily
- Number of juveniles: daily
- Stillborn juveniles and aborted eggs: daily
- Appearance of first brood
- Intrinsic rate of natural increase: test end
- Growth (total length and dry weight): test end


RANGE-FINDING STUDY
- Test concentrations: 0.1 - 1 - 10
Immobilization Rates in the Preliminary Test after 24 and 48 h of Exposure
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal Loading of the Water Accommodated Fraction (WAF)*
[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicate Replicate
1 2 MV 1 2 MV
10 0 0 0 0 0 0
1 0 0 0 0 0 0
0.1 0 0 0 10 0 5
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.048 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality of parent animals:
No immobilization or mortality of parent animals was observed, either in the control or in the water soluble fraction (WSF).
- No. of offspring produced per day per female:
In the control group and the water soluble fraction (WSF) five broods were released during the test period by all of the daphnids.
The mean number of offspring alive produced per parent animal surviving at the end of the test was 140 juveniles in the control group and 133 juveniles in the water soluble fraction (WSF).
The reproductive output of the water soluble fraction (WSF) was comparable to the control group and not statistically significant reduced when compared to the control group by MANN-WHITNEY Rank Sum Test.
- Body length and weight of parent animals:
The total length and the dry weight of all living parent animals at the water soluble fraction (WSF) and control were determined at the end of the test.
- Type and number of morphological abnormalities:
None
- Number of males and females (parental):
No males were observed in either the control or the test group during the test.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.78 (CI 1.46 - 2.18 mg/L)

Reported statistics and error estimates:

The NOEC and LOEC were determined directly from the results. Significant deviations were determined in comparison to the control group using statistical standard procedures as normality test, equal variance test; t-test and MANN-WHITNEY Rank Sum Test.
Statistical evaluation of the number of juveniles was carried out by using MANN-WHITNEY Rank Sum Test.
For the determination of significant deviations for the intrinsic rates of natural increase t-test (p = 0.05) was used.
The coefficients of variation around the mean number of living offspring produced per parent for the control and the water soluble fraction (WSF) were evaluated.

Number of Juveniles in the Control and Test Group after 21 d

Geom. Mean Meas. Conc. [mg/L]																					No.	Mean No. of Juveniles			Compared
	Number of Juveniles in Replicate No.																			Total	of			CV	versus
																				No.	Parents	per Parent			Control
																					prod. Juv.	producing Juveniles			Red.
	1	2		3		4		5		6		7		8		9		10		å	N	MV ± SD		[%]	[%]
0.0483	120	144		134		136		134		132		135		133		132		133		1333	10	133 ±	6	4	5
Control	129	117		142		154		132		150		142		130		151		148		1395	10	140 ±	12	9	--

Mortality [%] of the Adult Daphnids after 7, 14 and 21 d of Exposure

              (n = 10)

Geometric Mean Measured Concentration [mg/L]	Adult Mortality [%]
	7 d	14 d	21 d
0.0483	0	0	0
Control	0	0	0

First Appearance of Living Juveniles in the Individual Groups

Geometric Mean Measured Concentration [mg/L]	Day of First Brood										First
	in Replicate No.										Appearance
	1	2	3	4	5	6	7	8	9	10	Mean Day
0.0483	9	9	8	8	8	9	9	8	8	8	8.4
Control	8	8	8	8	9	8	8	8	8	8	8.1

Total Length and Dry Weight of the Parent Animals

Geometric Mean Measured Concentration [mg/L]	Total Length of the Parent Animals [mm]										MV	N	Dry Weight [mg]
	Replicate No.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
0.0483	5.25	5.00	5.75	5.50	5.50	5.50	5.50	5.25	5.50	5.25	5.40	10	8.6	0.86
Control	5.25	5.50	5.75	5.25	5.75	5.50	5.25	5.25	5.75	5.50	5.48	10	7.6	0.76

Validity criteria fulfilled:

yes

Conclusions:

In the water soluble fraction (WSF) of the test item Amines, bis (hydrogenated tallow alkyl) with a nominal loading concentration of 10.0 mg/L corresponding to a geometric mean measured concentration of 0.0483 mg/L, no statistically or biologically significant effects were observed. The No Observed Effect Concentration (NOEC) and the Lowest Observed Effect Concentration (LOEC) were assessed at:

NOEC: 0.0483 mg/L
LOEC: > 0.0483 mg/L

All effects given are based on the geometric mean measured concentration of the water soluble fraction (WSF) of the test item Amines, bis (hydrogenated tallow alkyl).

Executive summary:

The Daphnia magna Reproduction Test with Natural River Water (Semi-Static, 21 d) of the water soluble fraction (WSF) of the test item Amines, bis (hydrogenated tallow alkyl)(CAS No. 61789-79-5, batch number: S001020) was conducted as a limit test according to OECD 211 (2008) from 2010-03-22 to 2010-04-21, with the definitive exposure phase from 2010-03-24 to 2010-04-14, at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany .

Test system was Daphnia magna STRAUS (Clone 5). 10 test organisms, individually held, were used for the limit concentration and control. At the beginning of the test they were 2 to 24 h old. The test method was semi-static. The test solutions were renewed 3 times per week. Aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects on parameters such as adult mortality, intrinsic rate of natural increase, time of production of first brood, occurrence of aborted eggs and stillborn juveniles, dry body weight and length of the parent animals.

Amines, bis (hydrogenated tallow alkyl) (CAS No. 61789-79-5) is insoluble in water and has a strong tendency to adsorb to negatively charged surfaces such as suspended matter, algae and test vessels or organic material (including dissolved organic matter such as humic acids). Many cationic substances in general but long chain dialkyl amines in particular rank among the most difficult substances to test in environmental toxicology. Standard guideline studies are inappropriate to test substances with such properties and the current REACH Guidance Documents do not provide sufficient guidance concerning bioavailability and exposure assessment for cationic surface-active substances like the long chain dialkyl amines as these were written with normal hydrophobic chemicals in mind, failing to take into account the lack of bioavailability that occurs in the environment with these substances.

The aquatic ecotoxicity tests with long chain dialkyl amines were therefore performed in river water to allow a PEC_aquatic,bulk/PNEC_aquatic,bulkapproach 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, 2001).

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 to interpret, more environmental realistic, and if compared to PEC_bulkclearly provide a more appropriate assessment of risks for the environment.

The water soluble fraction (WSF) of the test item Amines, bis (hydrogenated tallow alkyl)with a nominal loading of 10 mg test item/L was tested as limit concentration. Prior to the preparation of the WSF, the river water was filtered using a 0.45 µm filter.

The limit concentration based on the results of a non GLP preliminary range finding test conducted as acute immobilization test with three water accommodated fractions under static conditions over a period of 48 h. The water soluble fraction (WSF) of the test item Amines, bis (hydrogenated tallow alkyl)and the control were analytically verified via LC-MS of samples on days 0, 5, 14, 16, 19 (fresh media, 0 h) and on days 2, 7, 16, 21 (old media, 48 h) and 19 (old media, 72 h). The sorption of the test item to the glassware was quantified once during the test period in a glass beaker, which has not been exposed before. The adsorbed fraction (day 19) corresponded to 57% of the initial concentration. Details of the analytical method are presented in part ‎10. All effect values are given based on the geometric mean measured concentration of the test item Amines, bis (hydrogenated tallow alkyl), determined to be: 0.0483 mg/L.

·   The average number of juveniles per parent in the control group was 140 and 133 in the water soluble fraction (WSF) of the test item after 21 days. The reproductive output of the water soluble fraction (WSF) was comparable to the control group and not statistically significant reduced when compared to the control group by Mann-Whitney Rank Sum Test (p = 0.05).

·   The coefficient of variation of the mean number of living offspring produced per parent in the control group was 9 % and 4 % in the water soluble fraction (WSF).

·   The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The mean IR of the surviving daphnids of water soluble fraction (WSF) was comparable to the control group.

·   No immobilization or mortality of parent animals occurred in the control or test groups.

·   No stillborn juveniles and aborted eggs were produced by the control group. Two aborted eggs were observed in the water soluble fraction (WSF).

·   The mean day of release of first brood was day 8.1 in the control and day 8.4 in the water soluble fraction (WSF). In the control as well as in the test groups the first brood occurred until day 9. In the control and the WSF five broods were released during the test period by all daphnids.

·   No malesand ephippia (winter eggs) occurred in control or test groups.

There was no statistically or biologically significant effect observed in the water soluble fraction (WSF) of the test item Amines, bis (hydrogenated tallow alkyl), when compared to the control.

Table 1:    Summary of all Test Item Related Effects

Based on the geometric mean measured concentration of the test item Amines, bis (hydrogenated tallow alkyl)

Effects	Amines, bis (hydrogenated tallow alkyl) Geometric mean measured concentration of the Test Item [mg/L]		Significance
	Control	0.0483
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)	140 ± 12	133 ± 6	no^1)
Mean Intrinsic Rate of Natural Increase	0.52	0.49	no^2)
Appearance of First Brood [Mean Day]	8.1	8.4	no^3)
Mean Number of Broods	5	5	no^3)
Stillborn Juveniles and Aborted Eggs	0	2	no^3)
Adult Mortality after 21 Days [%]	0	0	no^3)
NOECReproduction	0.0483 mg/L
LOECReproduction	> 0.0483 mg/L

SD    = Standard deviation

no     = No biological significance

¹⁾       = Statistically significant difference (t-test, p = 0.05), but biologically not significant

²⁾       = Statistically significant difference (Mann-Whitney Rank Sum Test, p = 0.05), but biologically not significant

³⁾       = Biologically not significant

 

·   Water quality parameters as pH-value, dissolved oxygen, water hardness and temperature were determined to be within the acceptable limits.

·          In order to prove the validity of the test system and test conditions at the test facility, an acute immobilization test according to DIN 38412 L 11 was carried out with potassium dichromateas reference item once per month.
The EC₅₀ of the reference item at 1.78 mg/L after 24 h was within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS P 9/2 (05/1996) 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.