Amines, N-(C18-unsatd. alkyl)trimethylenedi-, ethoxylated

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:

2009-09-02 to 2010-01-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP, higher tier test with river water applying bulk approach, incl. chemical analyses, all validity criteria fulfilled.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The long term toxicity test result as observed for the tallow based product is read across to the Oleyl based product. The products differ only in their alkylchain distribution. Both tallow and Oleyl are natural fats for which the composition slightly changes over time due to changes in the environmental considitions.

The tallow alkyl fat which is used for the production of CAS no 1268344-02-0 are for 99 % straight chain alkyl fats

Commercial tallow alkyl fats have the following distribution:

C12 about 2 %
C14 about 4 %
C16 about 31 %
C18 about 65 %,
where 40 –50 % are unsaturated.

The oleyl fate which is used for the production of CAS no 1290049-56-7 are also straight chain fates

Commercial Oleylfats have the following distribution:

C12 1 %
C14 2 - 4 %
C16 and C16’ 12 - 14 %
C18, C18’ and C18’’ > 80 % (C18: 8 %, C18’cis: 46 %, C18’trans: 21 %, C18’’: 5 %)
C20 1 %
(a single prime indicates one double bond, a double prime indicates two double bonds):

When the water solubility of cationic surfactants based on tallow are compared to oleyl based cationic surfactants it is consistently the tallow product which has a sligthly lower water solubility and due to that also a slightly lower bioavailability. This lower bioavailability regularly results is slight differences in biodegradation rate and ecotoxicity. The substances are however difficult to test substances and the variation in the test results is especially in ecotoxicity testing frequently larger than the difference in bioavailability. It is therefore considered justified to use the tallow product test results for read across for the oleyl based product.

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 30.0 and 270 µg/L and the control were analysed at least once within 7 days in the fresh (0 h) and old media (48 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 additional replicates without algae and test organisms were prepared and stored under test conditions.
The adsorption of the test item was quantified exemplarily. Therefore, two test replicates containing daphnids and algae of the test concentration 270 µg/L, containing daphnia and algae, were prepared analogously to the test design of the definitive test, emptied after 72 h of exposure and rinsed twice with demineralised water. Thereafter an extraction of the test vessel was carried out. The test item concentration in the fresh (0 h) and old media (72 h) were analytically verified, too.

- Sample storage conditions before analysis: All samples were stored at room temperature, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution (10 mg/L) was freshly prepared with natural river water. The stock solution was treated with ultrasound for 15 min at room temperature.
- Eluate: Natural river water
- Differential loading: 10.0 - 30.0 - 90.0 - 270 - 810 µg/L
- Controls: 10 replicates of natural water without test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none

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
[µg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Sept. 2nd Sept. 4th Sept. 9th Sept. 11th Sept. 16th Sept. 18th
810 344 297 n.a. n.a. n.a. n.a.
270 -- -- 335 312 308 317
Control 335 313 322 303 299 326

Test temperature:

Temperature [°C]
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Sept. 2nd Sept. 4th Sept. 9th Sept. 11th Sept. 16th Sept. 18th
810 20.3 21.2 n.a. n.a. n.a. n.a.
270 -- -- 21.5 21.5 21.9 21.8
Control 19.6 21.6 21.1 21.7 22.0 22.0

pH:

pH-Values
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Sept. 2nd Sept. 4th Sept. 9th Sept. 11th Sept. 16th Sept. 18th
810 8.00 8.23 n.a. n.a. n.a. n.a.
270 -- -- 7.92 8.05 7.72 7.96
Control 7.98 7.65 7.84 7.87 7.84 7.91

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Sept. 2nd Sept. 4th Sept. 9th Sept. 11th Sept. 16th Sept. 18th
810 9.48 9.16 n.a. n.a. n.a. n.a.
270 -- -- 9.74 7.65 9.13 6.51
Control 9.58 7.35 10.28 6.32 9.23 7.03

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, Germany. Specifications of the natural river water are given in the table below.
River: Leine
Location D-31171 Nordstemmen
Sampling Date 2009-05-18 2009-12-15
Dates of use (experimental phase)
2009-07-22 to 2009-08-12 2010-01-15 to 2010-01-18
(Definitive exposure phase) (Determination of Adsorption)
Weather conditions
on Day of Sampling Dry, few clouds, ca. 19 °C Cloudy, ca -1 °C
Colour Yellowish, slightly turbid Yellowish, clear
pH 7.94 7.97
Conductivity [µS/cm] 736 386
Dissolved Oxygen [mg O2/L] 9.02 8.62
DOC [mg C/L] 3.0 3.9
TOC [mg C/L] 3.1 3.9
Ammonium-N [mg N/L] 0.109 0.042
Nitrate-N [mg N/L] 3.43 2.62
Total Nitrogen [mg N/L] 5.23 3.53
o-Phosphate-p [mg P/L] 0.118 0.062
Total Phosphate [mg P/L] 0.141 0.053
Suspended Matter [mg/L] 11.7 16.2
Total Hardness [mg CO3/L] 322 154
Storage conditions 6 ± 2 °C < 10 °C
(Frozen due to low outdoor temperature)

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: 10 - 1 - 0.1 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 70 70 70 100 100 100
0.1 0 0 0 0 0 0
Control 0 0 0 0 0 0

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

270 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: adverse effect on reproduction

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

91.3 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

adult mortality

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

421 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

adult mortality

Remarks on result:

other: (256 - 785)

Details on results:

- Mortality of parent animals:
The test item induced significant adult mortality at the concentration levels 270 µg/L (30 %) and 810 µg/L (100 %) after 21 days. At the other concentration levels and in the control no significant mortality (≤ 20 %) of parent animals was observed.
The EC10 for adult mortality after 21 days was calculated to be 91.3 µg/L. The EC50 for adult mortality after 21 days was calculated to be 421 µg/L (CI: 256 - 785 µg/L).
- No. of offspring produced per day per female: Five broods were released by all surviving animals of the control and the test groups. Due to mortality of all parental daphnids at the concentration level 810 µg/L no reproduction was observed.
The mean number of offspring alive produced per parent animal surviving at the end of the test was 130 juveniles in the control group.
The reproductive output was statistically significant increased at the tested concentration level of 270 µg/L when compared to the control (One Way Analysis of Variance, DUNNETT’S method, p = 0.05). In this study no reduction of the reproductive output, but hormesis was observed.


- Body length and weight of parent animals: The mean dry body weight of the surviving parental daphnids at the tested concentration levels was comparable to the control group. The mean dry body weight at the concentration levels 10.0 to 270 µg/L ranged from 1.09 to 1.25 mg per daphnid. The mean dry body weight in the control was 1.12 mg per daphnid.
The mean values of the total body length at the tested concentrations were in the range of 5.53 to 5.75 mm per daphnid and comparable to the mean value of 5.43 mm per daphnid at the control group (One Way Analysis of Variance, p = 0.05).


- Type and number of morphological abnormalities: Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was ≤ 2 % at the concentration levels 10.0 to 270 µg/L, which is considered to be not biologically significant. At the control group only one stillborn juvenile and no aborted eggs were observed.
- 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 all test item and control groups producing juveniles was between day 8 and day 9

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.58 (CI 1.43 - 1.74)

Reported statistics and error estimates:

The NOEC value for the adult mortality as the most sensitive effect was determined directly from the observation data. 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, the intrinsic rates of natural increase and the body length was carried out using One Way Analysis of Variance, 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 EC50-value of the reference test was calculated by
EC-VALUES sigmoidal dose-response regression. The confidence interval for the EC50-value was calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism.
The EC50 for adult mortality was calculated as the geometric mean of the lowest concentration causing 100 % adult mortality (1000 µg/L) and the highest concentration causing 0 % adult mortality (320 µg/L), because only one partial effect was observed. These concentrations were also used as confidence limits.
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 effects on reproduction.

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

Nominal conc. [µg/L]												No.	Mean No. of Juveniles			Comparison
	Number of Juveniles in Replicate No.										Total	of			CV	versus
											No.	Parents	per Parent			Control
												prod. Juv.	producing Juveniles			Inc.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
810	--	--	--	--	--	--	--	--	--	--	--	0	n.a.		n.a.	n.a.	n.a.
270	--	198	160	185	195	--	--	169	146	155	1208	7	173 ±	20	12	33	yes
90.0	152	143	144	137	127	127	142	128	--	113	1213	9	135 ±	12	9	4	no
30.0	109	127	138	155	120	135	154	144	131	136	1349	10	135 ±	14	11	4	no
10.0	151	141	146	145	147	145	140	123	140	151	1429	10	143 ±	8	6	10	no
Control	136	110	129	136	144	154	127	138	117	112	1303	10	130 ±	14	11	—	—

Stillborn Juveniles and Aborted Eggs after 21 Days

Nominal concentration	Number of		Total No.		Mean Stillborn Juveniles and Aborted Eggs per Parent alive
	Stillborn	Aborted
[µg/L]	Juveniles	Eggs	å	N	MV ± SD
810	--	--	--	0	--
270	4	6	10	7	1.43 ± 1.62
90.0	7	17	24	9	2.67 ± 2.24
30.0	12	14	26	10	2.60 ± 2.27
10.0	6	4	10	10	1.00 ± 0.94
Control	1	0	1	10	0.10 ± 0.32

First Appearance of Living Juveniles in the Individual Groups

Nominal concentration	Day of First Appearance of Living Juveniles										First
	in Replicate No.										Appearance
[µg/L]	1	2	3	4	5	6	7	8	9	10	Mean Day
810	--	--	--	--	--	--	--	--	--	--	--
270	--	8	8	8	8	--	--	8	8	8	8.0
90.0	8	8	8	8	8	8	8	8	--	8	8.0
30.0	8	8	8	8	8	8	8	8	8	8	8.0
10.0	8	8	8	8	9	8	8	8	8	9	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 [%]
[µg/L]	7 days	14 days	21 days
810	100*	100*	100*
270	10	10	30*
90.0	0	10	10
30.0	0	0	0
10.0	0	0	0
Control	0	0	0

Total Body Length and Dry Weight of the Parent Animals

Nominal concentration [µg/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
810	--	--	--	--	--	--	--	--	--	--	--	0	--	--
270	--	6.00	5.25	5.75	5.25	--	--	6.00	6.00	6.00	5.75	7	8.2	1.17
90.0	5.50	6.00	5.50	5.50	5.50	5.75	5.50	5.75	--	5.25	5.58	9	10.7	1.19
30.0	5.75	5.75	5.50	5.50	5.25	5.75	5.50	5.25	5.75	5.25	5.53	10	10.9	1.09
10.0	5.50	5.50	5.75	6.00	5.25	6.00	5.75	5.25	5.75	5.50	5.63	10	12.5	1.25
Control	5.25	5.25	5.75	5.25	5.25	5.75	5.25	5.75	5.50	5.25	5.43	10	11.2	1.12

Validity criteria fulfilled:

yes

Conclusions:

The statistically significantly increased reproduction rate at the concentration level 270 µg/L was the most sensitive effect in this study. At this concentration level a biologically significant adult mortality of 30 % after 21 days was observed, too. In this study no reduction of the reproductive output, but hormesis was observed. Therefore, the NOEC was additionally assessed, taking hormesis not into account, to be the highest test concentration with no reduction of the reproductive output.
The EC10-value for the reproductive output was calculated by sigmoidal dose-response regression based on the nominal test item concentrations. An 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.

Effect values EC10, Reproduction : 130 µg/L (reproduction increase)
EC50, Reproduction : Not determinable

Adverse effect value NOECReproduction : 270 µg/L

The recoveries in the fresh media were in the range of 65 to 120 % of the nominal values. In the old media (after 48 h or 72 h) the recoveries decreased to values in the range of 44 to 99 %. Biodegradation as possible reason for this decline is very unlikely considering the short time frame between the refreshments of the test solutions. The concentration of the test item adsorbed to the glassware as determined exemplarily at the test concentration level of 270 µg/L was 23.9 µg/L which corresponds to 9 % of the nominal concentration. The observed concentration decrease between fresh and old media is thus only for a small fraction caused by sorption to the glassware. The main reduction is therefore most likely caused by thermodynamically more favourable redistribution of the sorbed fraction resulting in an additional sorption to suspended matter and DOC. Because of the limited sorption to glassware and unlikeliness of biodegradation as possible reason for the reduction of the observed concentration it must be concluded that the test organisms were fully exposed to the bulk concentration of the test substance during the test. All effect values are therefore 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 Tris (2-hydroxyethyl) oleyl diaminopropane (CAS no. 90367-27-4, batch number S001023) was conducted according to OECD 211 (2008) from 2009-09-02 to 2010-01-22 with the definitive exposure phase between 2009-09-02 to 2009-09-23 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 hours 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.

Tris(2-hydroxyethyl) oleyl diaminopropane (CAS No. 90367-27-4) is insoluble 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 ethoxylated diamines 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 ethoxylated diamines 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 the ethoxylated 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 Tris (2-hydroxyethyl) oleyl diaminopropane were selected after a preliminary acute immobilization test (48 h, static) as follows: 10.0 - 30.0 - 90.0 - 270 - 810 µg/L. The test item Tris (2-hydroxyethyl) oleyl diaminopropane was analytically verified in the concentration levels 30.0 and 270 µg/L and in the control via LC-MS/MS of samples taken on days 0, 7, 14, 16 (fresh media) and on days 2, 9, 16 (old media, 48 hours) and 19 (old media, 72 hours).
The recoveries in the fresh media were in the range of 65 to 120 % of the nominal values. In the old media (after 48 h or 72 h) the recoveries decreased to values in the range of 44 to 99 %.

Biodegradation as possible reason for this decline is very unlikely considering the short time frame between the refreshments of the test solutions. The concentration of the test item adsorbed to the glassware as determined exemplarily at the test concentration level of 270 µg/L was 23.9 µg/L which corresponds to 9 % of the nominal concentration. The observed concentration decrease between fresh and old media is thus only for a small fraction caused by sorption to the glassware. The main reduction is therefore most likely caused by thermodynamically more favourable redistribution of the sorbed fraction resulting in an additional sorption to suspended matter and DOC. Because of the limited sorption to glassware and unlikeliness of biodegradation as possible reason for the reduction of the observed concentration it must be concluded that the test organisms were fully exposed to the bulk concentration of the test substance during the test. All effect values are therefore given are based on the nominal test item concentrations.

·   The average number of juveniles per parent in the control group was 130 after 21 days. The reproductive output was statistically significant increased at the tested concentration level of 270 µg/L when compared to the control (One Way Analysis of Variance, Dunnett’smethod, p = 0.05). Due to mortality of all parental daphnids at the concentration level 810 µg/L no reproduction was observed. In this study no reduction of the reproductive output, but hormesis was observed. The EC₁₀-value for the increase of the reproductive output was calculated by sigmoidal dose-response regression to be 130 µg/L. An 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. As adverse effects the NOEC was assessed to be 270 µg/L (the highest test concentration with no reduction of the reproductive output).

·   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 used for calculation of population growth and maintenance. The mean IR of the surviving daphnids of the treatment groups were compared to the control by One Way Analysis of Variance (p = 0.05). There was no statistically significant difference observed at any of the concentration levels.

·   Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles (sum of stillborn juvenile and aborted eggs) were ≤ 2 % at all the test and control groups, which is not biologically significant.

The mean day of release of the first brood was day 8.0 in the control group. In the tested concentration levels 30.0 to 270 µg/L the mean day of release of the first brood was comparable to the control group. The first brood was released until day 9 by all surviving daphnids of the control group and the tested concentration levels 10.0 to 270 µg/L. Five broods were released by all surviving animals of the control and the test groups.

The reproduction rate and the statistically significant increase of 33 % in comparison to the control at the concentration level 270 µg/L was the most sensitive effect in this study.
A summary of all test item related effects on reproduction is given in Table 1. An EC₅₀-value for the reproductive output was not determinable because no effects ≥ 50 % (reduction or increase of the reproductive output) occurred within the tested concentration range. The NOEC was assessed as adverse effect level directly from the observation data taking the observed hormesis not into account.

Table 1:        Test Item Related Effects on Reproduction, NOEC and EC₅₀

Effects	Nominal Concentration [µg/L]
	Control	10.0	30.0	90.0	270	810
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)	130 ± 14	143 ± 8	135 ± 14	135 ± 12	173 ± 20^1)	--
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent	11	6	11	9	12	--
Mean Intrinsic Rates of Natural Increase	0.50	0.50	0.49	0.50	0.52	--
Appearance of First Brood [Mean Day]	8.0	8.2	8.0	8.0	8.0	--
Number of Broods	5	5	5	5	5	--
EC50, Reproduction with Confidence Interval (CI) p = 95 %	Not determinable (CI: Not applicable)
NOEC (adverse effect)	270 µg/L^2)

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 270 µg/L (30 %) and 810 µg/L (100 %) after 21 days. At the other concentration levels and in the control no significant mortality (≤ 20 %) of parent animals was observed. The EC₁₀ for adult mortality after 21 days was calculated to be 91.3 µg/L. The EC₅₀ for adult mortality after 21 days was calculated to be 421 µg/L (CI: 256 - 789 µg/L).

·   The mean dry body weight and mean total body length of the surviving parental daphnids at the tested concentration levels were comparable to the control group.
The mean dry body weight at the concentration levels 10.0 to 270 µg/L ranged from 1.09 to 1.25 mg per daphnid. The mean dry body weight in the control was 1.12 mg per daphnid.
The mean values of the total body length at the tested concentrations were in the range of 5.53 to 5.75 mm per daphnid and comparable to the mean value of 5.43 mm per daphnid at the control group (One Way Analysis of Variance, p = 0.05).

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

Effects	Nominal Concentration [µg/L]
	Control	10.0	30.0	90.0	270	810
Adult Mortality after 21 Days [%]	0	0	0	10	30^1)	100^1)
Parent Animals: Mean Dry Weight [mg]	1.12	1.25	1.09	1.19	1.17	--
Parent Animals: Mean Body Length [mm]	5.43	5.63	5.53	5.58	5.75	--
EC10, Adult Mortality	91.3 µg/L
EC50, Adult Mortality with Confidence Interval (CI) p = 95 %	421 µg/L (CI: 256 - 785 µg/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 1.58 mg/L after 24 hours 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

One long term toxicity test with daphnia has been performed in accordance with OECD 211. The NOEC for reproduction is 270 µg/L. The EC10 and EC50 for parental mortality are resp. 91.3  and 421 µg/L. The effects are expressed as nominal values because the tests were performed with river water as they are intended to be used in an evaluation of the environmental risks based on the Bulk approach.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

91.3 µg/L

Additional information

As a worst-case the EC10 for parental mortality of 91.3 µg/L is used for risk assessment purposes and not the NOEC of 270 µg/L for reproduction.