Amides, from C8-10-fatty acids and tetraethylenepentamine

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2010-02-22 to 2010-03-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP, All validity criteria fulfilled, complete identification of test substance, including chemical analyses

Justification for type of information:

Refer to read across justification document in section 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Two concentration levels 0.320 and 0.800 mg/L and the control were analysed at least once within 7 days in the fresh (0 h) and old media (48 h). Due to the mortality of all parent animals at concentration level 2.00 mg/L on day 2, samples of the concentration level 0.800 mg/L were analysed instead at the remaining sampling intervals.
- 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 additional replicates without algae and test organisms were prepared and stored under test conditions.
The sorption of the test item to the glassware was quantified once during the test period after the first sampling interval. Therefore, one test vessel of the test concentration 0.320 mg/L, containing daphnia and algae, was emptied after 72 h of exposure and rinsed twice with water. Thereafter an extraction of the test vessel was carried out.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg/L was freshly prepared with dilution water and treated with ultrasound for 5 min at room temperature.
- Eluate: Natural river water
- Differential loading: 0.128 - 0.320 - 0.800 - 2.00 - 5.00 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:

no

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Hardness:

Total Hardness [mg/L] as CaCO3
Nominal
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 18 Day 21
Feb. 22nd Feb. 24th March 1st March 3rd March 12th March 15th
5.00 114 114 n.a. n.a. n.a. n.a.
0.800 -- -- 122 122 119 115
Control 100 114 76 116 112 119

Test temperature:

Temperature [°C]
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 18 Day 21
Feb. 22nd Feb. 24th March 1st March 3rd March 12th March 15th
5.00 22.0 21.3 n.a. n.a. n.a. n.a.
0.800 -- -- 21.3 21.8 19.2 21.7
Control 19.1 21.5 19.0 21.7 19.0 21.9

pH:

pH-Values
Nominal
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 18 Day 21
Feb. 22nd Feb. 24th March 1st March 3rd March 12th March 15th
5.00 7.75 7.61 n.a. n.a. n.a. n.a.
0.800 -- -- 7.84 7.62 8.16 7.78
Control 8.07 7.79 8.07 7.70 8.11 7.59

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 18 Day 21
Feb. 22nd Feb. 24th March 1st March 3rd March 12th March 15th
5.00 8.44 8.42 n.a. n.a. n.a. n.a.
0.800 -- -- 8.89 7.43 9.48 8.67
Control 9.32 6.95 9.41 7.98 9.60 7.90

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to information on materials and methods

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, Germany. Specifications of the natural river water are given in the table below.
River: Leine
Location D-31171 Nordstemmen
Sampling Date 2009-12-15
Dates of use
(experimental phase) 2010-01-15 to 2010-01-18

Weather conditions
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 - 10 mg/L
Immobilization Rates [%] of the Preliminary Acute Immobilization Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item
Concentration
[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicate Replicate
1 2 MV 1 2 MV
10 100 100 100 100 100 100
1 0 0 0 10 10 10
0.1 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

320 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

712 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Parental

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.775 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: (0.320-2.00)

Details on results:

- Mortality of parent animals:
The test item induced significant adult mortality at the concentration levels 0.800 mg/L (70 %), 2.00 and 5.00 mg/L (100 %) after 21 days. At the other concentration levels and in the control no significant mortality (≤ 20 %) of parent animals was observed. The EC50 for adult mortality after 21 days was calculated respectively to be 0.775 mg/L (95 % confidence limits: 0.320- 2.00 mg/L).
- No. of offspring produced per day per female:
Four to six broods were released by all surviving animals of the control and concentration levels 0.128 to 0.800 mg/L.
The average number of juveniles per parent in the control group was 121 after 21 days. The reproductive output was statistically significant increased at the tested concentration level 0.320 mg/L and decreased at 0.800 mg/L when compared to the control (One Way Analysis of Variance, DUNNETT´S method p = 0.05). The reproductive output at the concentration level 0.128 mg/L was comparable to the control group.
In this study hormesis was observed at the concentration level 0.320 mg/L.
An EC10- and EC50-value for the reproductive output could not be calculated, because no effects ≥ 50 % (reduction or increase of the reproductive output) occurred within the tested concentration range and the effects were not concentration related. As adverse effects the NOEC was assessed to be 0.320 mg/L (the highest test concentration with no reduction of the reproductive output).
- Body length and weight of parent animals:
The mean values of the dry body weight of the surviving parental daphnids, determined at the end of the study, at the concentration levels 0.128 to 0.320 mg/L were in the range of 0.70 to 0.96 mg and comparable to the mean value of 0.91 mm per daphnid at the control group. At the concentration level 0.800 mg/L the mean value of the dry body weight was 0.10 mg.
The mean values of the total body length of the surviving parental daphnids at the concentration levels 0.128 to 0.800 mg/L were in the range of 5.00 to 5.65 mm per daphnid and comparable to the mean value of 5.55 mm per daphnid at the control group.
- Number of males and females (parental) No males were observed in either the control or the test groups during the test.
- Time to first brood release or time to hatch:The first day of appearance of juveniles at the test item concentration levels 0.128 and 0.320 mg/L and control groups producing juveniles was between day 7 and day 9. At the tested concentration 0.800 mg/L the first appearance of juveniles was belated on days 10 and 12 at 2 of 3 surviving parental daphnids, which is regarded to be biologically noticeable

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.44 (CI 1.35 - 1.54)

Reported statistics and error estimates:

Significant deviations were evaluated in comparison to the control using statistical standard procedures as Normality Test, Equal Variance Test and Analysis of Variance. Statistical evaluation of the reproduction rates and of the intrinsic rates of natural increase was carried out using One Way Analysis of Variance, DUNETT´S method (p = 0.05). The coefficients of variation around the mean number of living offspring produced per parent in the control and the test groups were evaluated.
The estimation of the EC10- and EC50 -value for adult mortality and the EC50 –value for the reference item was carried out by using sigmoidal dose-response regression. The confidence intervals for the EC50-value of the reference item were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism. For the EC50-value of adult mortality the highest concentration causing no effect and the lowest concentration producing 100 % were used as confidence limits. An EC10- or EC50-value for the reproductive output could not be calculated, because no effects ≥ 50 % (reduction or increase of the reproductive output) occurred within the tested concentration range and the effects were not concentration related.

Number of Juveniles alive in the Control and Test Groups after 21 Days

Nominal conc. [mg/L]												No.	Mean No. of Juveniles alive			Comparison
	Number of Juveniles alive in Replicate No.										Total	of			CV	versus
											No.	Parents	per Parent			Control
												prod. Juv.	producing Juveniles			Red.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
5.00	--	--	--	--	--	--	--	--	--	--	--	0	n.a.		n.a.	n.a.	n.a.
2.00	--	--	--	--	--	--	--	--	--	--	--	0	n.a.		n.a.	n.a.	n.a.
0.800	--	98	--	--	--	--	87	--	--	97	282	3	94.0 ±	6	6	22.3	yes
0.320	151	150	130	147	162	125	149	160	137	145	1456	10	146   ±	12	8	-20.7	yes
0.128	124	103	106	120	101	116	114	104	102	133	1123	10	112   ±	11	10	7.44	no
Control	95	150	118	126	118	118	117	128	125	112	1207	10	121   ±	14	11	—	—

First Appearance of Living Juveniles at the Individual Groups

Nominal concentration	Day of First Appearance of Living Juveniles										First
	in Replicate No.										Appearance
[mg/L]	1	2	3	4	5	6	7	8	9	10	Mean Day
2.00	--	--	--	--	--	--	--	--	--	--	--
5.00	--	--	--	--	--	--	--	--	--	--	--
0.800	--	12*	--	--	--	--	10*	--	--	7	9.7
0.320	8	8	8	8	7	8	7	7	8	8	7.7
0.128	8	8	8	9	9	8	8	8	8	8	8.2
Control	8	8	8	8	8	8	8	8	8	8	8.0

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

                 (n = 10)

Nominal concentration	Adult Mortality [%]
[mg/L]	7 days	14 days	21 days
5.00	100*	100*	100*
2.00	100*	100*	100*
0.800	10	10	70*
0.320	0	0	0
0.128	0	0	0
Control	0	0	0

Total Body Length and Dry Weight of the Parent Animals

Nominal 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
2.00	--	--	--	--	--	--	--	--	--	--	--	0	--	--
5.00	--	--	--	--	--	--	--	--	--	--	--	0	--	--
0.800	--	4.75	--	--	--	--	5.00	--	--	5.25	5.00	3	0.3	0.10
0.320	5.75	5.50	5.50	5.75	5.75	5.75	5.75	5.75	5.25	5.75	5.65	10	9.6	0.96
0.128	5.50	5.75	5.25	5.50	5.00	5.25	5.75	5.25	5.25	5.50	5.40	10	7.0	0.70
Control	5.75	5.50	5.75	5.50	5.50	5.75	5.50	5.25	5.50	5.50	5.55	10	9.1	0.91

Validity criteria fulfilled:

yes

Conclusions:

The adult mortality at the concentration levels 0.800 and 2.00 mg/L was the most sensitive effect in this study. In this study hormesis was observed at the concentration level 0.320 mg/L. The reproductive output was statistically significantly decreased at 0.800 mg/L. The NOEC was assessed as adverse effect level directly from the observation data taking the observed hormesis not into account.
The estimation of an EC10- or EC50-value for the reduction or increase of the reproductive output could not be performed due to the absence of statistically significant effects on the reproduction. Effect values are as following:

Adverse effect value: NOECReproduction : 0.320 mg/L

Effect values: EC10, Reproduction : Not applicable
EC50, Reproduction : Not applicable

EC10, Adult Mortality : 0.712 mg/L
EC50, Adult Mortality : 0.775 mg/L (confidence limits: 0.320 – 2.00 mg/L)


The recoveries in the fresh media were in the range of 94 to 119 % of the nominal values. In the old media (after 48 h or 72 h) the recoveries decreased to values in the range of 66 to 123 %. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame between the refreshments of the test solutions. The adsorption of the test item to the glassware was determined at the concentration level 0.320 mg/L on day 21 to be < LOQ, which corresponds to < 5 % of the nominal concentration. The limited concentration decrease between fresh and old media is therefore most likely caused by thermodynamically more favourable redistribution of the sorbed fraction resulting in an additional sorption to suspended matter and DOC. Furthermore imidazolines like the test item have the potential to hydrolyze but is expected to be negligible under the applied test conditions. The results of the chemical analyses show that the test organisms were fully exposed to the test substance during the test. Therefore, all effect values given are based on the nominal test item concentrations.

Executive summary:

The Daphnia magna Reproduction Test with Natural River Water (Semi-Static, 21 d) of the test item Tall oil reaction products with tetraethylenepentamine (CAS no. 68555-22-6, batch number S000925) was conducted according to OECD 211 (2008) from 2010-02-22 to 2010-03-17 with the definitive exposure phase from 2010-02-22 to 2010-03-15, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str. 1, D-31157 , .

Test system was Daphnia magna STRAUS (Clone 5). 10 test organisms, individually held, were used per concentration level 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.

Talloil reaction products with tetraethylenepentamine (CAS No. 68555-22-6) is emulsifiable in water and also 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 amido amines/imidazolines 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 amido amines/imidazolines 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 amido amines/imidazolines 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 bulkapproach, 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_bulk clearly provide a more appropriate assessment of risks for the environment.

Nominal concentrations of the test item Tall oil reaction products with tetraethylenepentamine were selected after a preliminary acute immobilization test (48 h, static) as follows: 0.128 - 0.320 - 0.800 - 2.00 - 5.00 mg/L. The test item Tall oil reaction products with tetraethylenepentaminewas analytically verified in the concentration levels 0.320 and 2.00 mg/L and in the control via LC-MS/MS of samples taken on days 0 (fresh media) and 2 (old media, 48 h). Due to the mortality of all parent animals in concentration level 2.00 mg/L on day 2, the concentration level 0.800 mg/L was analysed instead. At the remaining sampling intervals the concentration levels 0.320 and 0.800 mg/L and the control were analytically verified of samples taken on day 7, 18 (fresh media) and day 9 (old media; 48h) and 21 (old media, 72 h).
The recoveries in the fresh media were in the range of 94 to 119 % of the nominal values. In the old media (after 48 h or 72 h) the recoveries decreased to values in the range of 66 to 123 %. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame between the refreshments of the test solutions. No adsorption ( LOQ; day 21) of the test item to the glassware was observed. The adsorbed fraction of the test item concentration was determined to be 5 % of the nominal concentration at the concentration level 0.320 mg/L. The limited concentration decrease between fresh and old media is therefore most likely caused by thermodynamically more favourable redistribution of the sorbed fraction resulting in an additional sorption to suspended matter and DOC. Furthermore imidazolines like the test item have the potential to hydrolyze but is expected to be negligible under the applied test conditions. The results of the chemical analyses show that the test organisms were fully exposed to the test substance during the test. Therefore, all effect values given are based on the nominal test item concentrations.

The average number of juveniles per parent in the control group was 121 after 21 days. The reproductive output was statistically significant increased at the concentration level 0.320 mg/L and statistically significant decreased at the concentration level 0.800 mg/L when compared to the control (One Way Analysis of Variance,Dunnett´s method p = 0.05). In this study hormesis at the concentration level 0.320 mg/L was observed. Due to mortality of all parental daphnids at the concentration levels 2.00 and 5.00 mg/L no reproduction was observed.

The coefficient of variation of the mean number of living offspring produced per parent alive in the control group was 11 %.

The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and number of offspring were An EC₁₀- or EC₅₀-value for the reproductive output could not be calculated, because no effects ≥ 50 % (reduction or increase of the reproductive output) occurred within the tested concentration range and the effects were not concentration related.used for calculation of population growth and maintenance. The IR of the surviving daphnids of the treatment groups were compared to the control (One Way Analysis of Variance, Dunnett´s method p = 0.05). There is a statistical significant difference determined at the concentration level 0.800 mg/L. The IR at the concentration level 0.128 and 0.320 mg/L were comparable to the control group. At the concentration levels 2.00 and 5.00 mg/L no IR was calculated due to mortality of all parental daphnids.

Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles (sum of stillborn juvenile and aborted eggs) was 2 % at the tested concentration level 0.128 mg/L and 1 % at the tested concentration level 0.320 mg/L. At the concentration level 0.800 mg/L the percentage of dead juveniles was 10%. At the control no dead juveniles or aborted eggs were observed.

The reduction of the reproductive output and the intrinsic rates of natural increase of the surviving parent animals at the concentration level 0.800 mg/L were the most sensitive effect in this study. A summary of all test item related effects on reproduction are presented in Table 1.

Table 1:        Test Item Related Effects on Reproduction and EC₅₀

Effects	Nominal Concentration [mg/L]
	Control	0.128	0.320	0.800	2.00	5.00
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)	121 ± 14	112 ± 11	146 ± 12^1)	94.0 ± 6^1)	--	--
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent	11	10	8	6	--	--
Number of Dead Juveniles and Aborted Eggs	0	19	18	30	--	--
Mean Intrinsic Rates of Natural Increase	0.49	0.48	0.51	0.43^1)	--	--
Appearance of First Brood [Mean Day]	8.0	8.2	7.7	9.7	--	--
Mean Number of broods	5.0	4.9	5.0	5.0	--	--
NOEC (adverse effect)	0.320 mg/L
EC50, Reproduction with Confidence Interval (CI) p = 95 %	Not determinable (CI: Not applicable)

SD    = Standard deviation

¹⁾       = Statistically significant (One Way Analysis of Variance, Dunnett’s method, p = 0.05)

--       = Not applicable due to the mortality of all parental animals

A summary of test item related effects concerning the adult mortality and growth (weight, length) is presented in Table 2.

The test item induced significant adult mortality at the concentration levels 0.800 mg/L (70 %), 2.00 and 5.00 mg/L (100 %) after 21 days. At the other concentration levels and at the control no significant mortality (≤ 20 %) of parent animals was observed. The EC₅₀ for adult mortality after 21 days was calculated respectively to be 0.775 mg/L (95 % confidence limits: 0.320- 2.00 mg/L).

The mean values of the dry body weight of the surviving parental daphnids, determined at the end of the study, at the concentration levels 0.128 to 0.320 mg/L were in the range of 0.70 to 0.96 mg and comparable to the mean value of 0.91 mm per daphnid at the control group. At the concentration level 0.800 mg/L the mean value of the dry body weight was 0.10 mg.
The mean values of the total body lengthof the surviving parental daphnids at the concentration levels 0.128 to 0.800 mg/L were in the range of 5.00 to 5.65 mm per daphnid and comparable to the mean value of 5.55 mm per daphnid at the control group.

Table 2:    Test Item Related Effects on Adult Mortality (EC₁₀, EC₅₀) and Growth (Weight, Length)

Effects	Nominal Concentration [mg/L]
	Control	0.128	0.320	0.800	2.00	5.00
Adult Mortality after 21 Days [%]	0	0	0	70^1)	100^1)	100^1)
Parent Animals: Mean Dry Weight [mg]	0.91	0.70	0.96	0.10	--	--
Parent Animals: Mean Body Length [mm]	5.55	5.40	5.65	5.00	--	--
EC10, Adult Mortality	0.712 mg/L
EC50, Adult Mortality with Confidence Interval (CI) p = 95 %	0.775 mg/L (CI: 0.320 – 2.00 mg/L)

¹⁾       = Mortality > 20 % biologically significant

--       = Not applicable due to 100 % mortality of all parental animals

·       No males and ephippia (winter eggs) were observed at the control or test groups.

 

·   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 1.44 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.

Description of key information

Read-across from structural analogue 68555 -22 -6

One long term toxicity test with daphnia has been performed. The 21 d EC10  for reproduction is 320 µg/L based on the nomimal test substance concentration.  Corrected for active ingredient which is 96% the EC10 is reduced to 307.2 µg/L..

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

307.2 µg/L

Additional information

One long term toxicity test with daphnia has been performed. The 21 d EC10 for reproduction is 320 µg/L based on the nomimal test substance concentration. Corrected for active ingredient which is 96% the EC10 is reduced to 307.2 µg/L. The test was performed with river water as it is intended to be used in an evaluation of the environmental risks based on the Bulk approach where it is essential to show that the organisms were fully exposed to the nominal test concentration. During the test a decrease of the concentration is observed of about 30%. Imidazolines are susceptible to hydrolysis and the decrease observed could therefore be assigned to either hydrolysis or sorption or even both processes. Imidazolines will however only hydrolyze to an amidoamine which is relatively resistant to hydrolysis under ambient conditions. As this hydrolysis will also occur under environmental conditions and amidoamines have a comparable ecotoxicity as the imidazolines the Nominal concentration based on active ingredient is considered as the concentration to be used for the dose effect assesment as no significant adsorption to glassware was observed and the organisms were thus fully exposed the the nominal concentration based on active ingredient. Initially to the imidazoline which have gradually changed into an amidoamine during the test. It is considered realistic to relate the observed effects to the nominal values based on active ingredient.