Amines, N-(3-aminopropyl)-N’-[3-(C16-18 (even numbered) and C18 unsaturated alkyl amino)propyl]trimethylenedi- and amines, N-(3-aminopropyl)-N’-(C16-18 (even numbered) and C18 unsaturated alkyl)trimethylenedi-

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-07-22 to 2010-01-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Amines, N-(3-aminopropyl)-N’-[3-(C16-18 (even numbered) and C18 unsaturated alkyl amino)propyl]trimethylenedi- and amines, N-(3-aminopropyl)-N’-(C16-18 (even numbered) and C18 unsaturated alkyl)trimethylenedi-

EC Number:

701-390-1

Molecular formula:

UVCB - No molecular formula; Avg mw = 390.26 mw C16 Tetramine = 412.751 mw C18 Tetramine = 440.805

IUPAC Name:

Amines, N-(3-aminopropyl)-N’-[3-(C16-18 (even numbered) and C18 unsaturated alkyl amino)propyl]trimethylenedi- and amines, N-(3-aminopropyl)-N’-(C16-18 (even numbered) and C18 unsaturated alkyl)trimethylenedi-

Constituent 2

Reference substance name:

68911-79-5

Cas Number:

68911-79-5

IUPAC Name:

68911-79-5

Details on test material:

- Name of test material (as cited in study report): Tallowtripropylenetetramine (CAS No. 68911-79-5)
- Substance type: Emulsifier
- Physical state: Solid, paste, pale yellow
- Composition of test material, percentage of components: Primary fatty amine: 3.1 (area %); Di amine: 11.3 (area %); Tri amine: 36.1 (area %); Tetra amine: 43.4 (area %); Penta amine: 6.1 (area %)
- Purity test date: 2009-02-05
- Lot/batch No.: S001255
- Expiration date of the lot/batch: 2018-01-23
- Storage condition of test material: 6 +/- 2 °C, protected from moisture and light, stored under nitrogen gas

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Two concentration levels 0.320 and 2.00 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 3 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 separate replicates without algae and test organisms were prepared and stored under test conditions.
Additionally, the freshly prepared stock solution of 10 mg/L was analytically verified at least once within 7 days to check their correct preparation. The analytical method was validated.
The adsorption of the test item was quantified exemplarily. Therefore, two test replicates containing daphnids and algae of the test concentration 0.800 mg/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. The analytical method was validated.
- Sample storage conditions before analysis: All samples were stored at room temperature if necessary

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (10 mg/L) was freshly prepared with natural river water for each water renewal. The stock solution was treated with ultrasound for 30 min at 40 °C.
- Eluate: Natural river water
- Differential loading: 0.128 - 0.320 - 0.800 - 2.00 - 5.00 mg/L (factor 2.5)
- Controls: 10 replicates of natural water without test item.

Test organisms

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

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Test conditions

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 14 Day 16
July 22nd July 24th July 29th July 31st August 5th August 7th
5.00 308 287 n.a. n.a. n.a. n.a.
0.800 -- -- 324 306 349 333
Control 313 290 308 290 345 338

Test temperature:

Temperature [°C]
Nominal
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
July 22nd July 24th July 29th July 31st August 5th August 7th
5.00 22.0 21.9 n.a. n.a. n.a. n.a.
0.800 -- -- 21.6 21.8 21.9 21.5
Control 20.6 22.0 21.7 21.8 21.4 21.4

pH:

pH-Values
Nominal
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
July 22nd July 24th July 29th July 31st August 5th August 7th
5.00 8.11 8.36 n.a. n.a. n.a. n.a.
0.800 -- -- 8.10 8.21 8.01 7.88
Control 7.92 8.20 8.04 8.10 7.98 7.89

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 14 Day 16
July 22nd July 24th July 29th July 31st August 5th August 7th
5.00 8.42 9.00 n.a. n.a. n.a. n.a.
0.800 -- -- 8.17 7.43 9.24 6.78
Control 8.73 7.40 8.30 7.68 9.22 7.19

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 2009-05-18 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: 1 - 10 mg/L
mmobilization 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 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

Adverse effect

Effect concentrations 2

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.974 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: (0.819 - 1.16)

Effect concentrations 3

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.712 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality of parent animals:
The test item induced significant adult mortality of 100 % at the tested concentration levels 2.00 and 5.00 mg/L 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 by sigmoidal dose-response regression to be 0.712 mg/L. The EC50 was calculated by sigmoidal dose-response regression to be 0.974 mg/L (CI: 0.819 - 1.16 mg/L).
- No. of offspring produced per day per female:
The number of juveniles in all replicates of control and test groups was recorded every day.
Five broods were released by all surviving animals of the control and the test groups. At the concentration levels 2.00 and 5.00 mg/L no juveniles occurred due to mortality of all daphnids.
The mean number of offspring alive produced per parent animal surviving at the end of the test was 144 juveniles in the control group.
The reproductive output was not statistically significant increased or reduced at any of the tested concentration levels when compared to the control (One Way Analysis of Variance, p = 0.05). An EC10- or EC50-value for the reproductive output was not determinable because no significant effects (reduction or increase of the reproductive output) occurred within the tested concentration range For the mean number of living offsprings produced per parent in the control group the coefficient of variation amounts to 5 % (Table 4). At the test groups the coefficient of variation came to a maximum of 14 % at the concentration level of 0.800 mg/L.
The mean number of juveniles of the surviving daphnids of the treatment groups were compared to the control by One Way Analysis of Variance, p = 0.05. The reproductive output was not statistically significant increased or reduced at any of the tested concentration levels when compared to the control (One Way Analysis of Variance, p = 0.05). An EC10- or EC50-value for the reproductive output was not determinable because no significant effects (reduction or increase of the reproductive output) occurred within the tested concentration range.

- Body length and weight of parent animals:
At the end of the test the total body length and the dry weight of all surviving parent animals at each tested concentration level and control were determined.
The mean dry body weight in the concentration levels 0.128 and 0.320 mg/L was determined to be 1.26 and 1.28 mg per daphnid. The mean dry body weight in the control was 1.39 mg per daphnid. At the concentration level 0.800 mg/L the mean dry body weight was biologically significantly reduced to 0.94 mg per daphnid, when compared to the control (reduction: 32 %).
The mean value of the total body length in the concentration levels 0.128 and 0.320 mg/L were determined to be 5.67 and 5.81 mm per daphnid and 5.86 mm per daphnid in the control group. At the concentration level 0.800 mg/L the mean dry body weight was statistically significantly reduced to 5.28 mm per daphnid. The total body length of the concentration levels 0.128 to 0.800 mg/L were compared to the control using KRUSKAL-WALLIS One Way Analysis of Variance on Ranks (p = 0.05), because normality test failed.
The most sensitive effect in this study was the reduction of the mean dry body weight and mean total body length of the parent daphnids at the concentration level 0.800 mg/L. At the concentration levels ≤ 0.320 mg/L no effects 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: please refer to the respective table

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.29 (CI 1.15 - 1.45)

Reported statistics and error estimates:

The NOEC and LOEC for the reproduction and the adult mortality as the most sensitive effects were determined directly from the observation data. Significant deviations were determined in comparison to the solvent control using statistical standard procedures as Normality Test, Equal Variance Test and Analysis of Variance.
For the determination of significant deviations for the reproduction rates, the intrinsic rates of natural increase and the body length One Way Analysis of Variance, DUNNETT’S method, p = 0.05 was used. Statistical evaluation of age of first reproduction and occurrence of stillborn juveniles and aborted eggs could not be carried out because Normality Test failed. The coefficients of variation around the mean number of living offspring produced per parent in the control, the solvent control and the test groups were evaluated.
EC50- values of the adult mortality and the reference item were calculated by sigmoidal dose-response regression. The confidence interval for the EC50 of the reference item was calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism. For the confidence interval of the EC50-value of the adult mortality the highest concentration causing no effect and the lowest concentration with 100 % adult mortality were used as confidence limits, because only one partial effect was observed. The EC50- value of the reproductive output was determined directly from the observation data because reduction of the reproductive output ≥ 50 % was not observed within the tested concentration range.

Any other information on results incl. tables

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

Nominal conc. [mg/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		[%]	[%]
5.00	--	--	--	--	--	--	--	--	--	--	--	0	n.a.		n.a.	n.a.	n.a.
2.00	--	--	--	--	--	--	--	--	--	--	--	0	n.a.		n.a.	n.a.	n.a.
0.800	152	142	142	174	158	--	117	148	116	--	1149	8	144 ±	20	14	0	no
0.320	155	152	156	178	148	162	170	158	--	157	1436	9	160 ±	9	6	11	no
0.128	153	145	162	--	144	156	131	163	137	116	1307	9	145 ±	15	11	1	no
Control	149	150	146	--	144	149	138	129	150	141	1296	9	144 ±	7	5	—	—

Nominal conc.   = Nominal concentration

N                          = Number of parents producing juveniles alive

MV ± SD              = Mean value ± standard deviation

CV                       = Coefficient of variation

Inc.                      = Increase on reproductive output in comparison to the control group

Stat.                     = Statistical significance (One Way Analysis of Variance, p = 0.05)

no                        = Statistically not significant

--                          = Not applicable, due to mortality of the parental animal

n.a.                      = Not applicable, due to the mortality of all daphnids

First Appearance of Living Juveniles in 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
5.00	--	--	--	--	--	--	--	--	--	--	--
2.00	--	--	--	--	--	--	--	--	--	--	--
0.800	8	8	8	8	8	--	8	9	8	--	8.1
0.320	8	8	7	8	8	7	8	8	--	8	7.8
0.128	7	8	8	--	8	8	8	8	7	7	7.7
Control	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	20
0.320	0	0	10
0.128	0	10	10
Control	0	0	10

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
5.00	--	--	--	--	--	--	--	--	--	--	--	0	--	--
2.00	--	--	--	--	--	--	--	--	--	--	--	0	--	--
0.800	5.25	5.00	5.25	5.25	5.50	--	5.50	5.25	5.25	--	5.28^1)	8	7.5	0.94^2)
0.320	6.00	5.75	5.50	6.00	5.50	6.00	5.50	6.00	--	6.00	5.81	9	11.5	1.28
0.128	5.75	5.75	5.75	--	5.50	5.50	5.75	6.00	5.75	5.25	5.67	9	11.3	1.26
Control	6.00	6.00	6.00	--	5.75	6.00	6.00	5.50	5.75	5.75	5.86	9	12.5	1.39

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

A statistically significant reduction or increase of the reproduction rate in comparison to the control group was not observed in the tested concentration range. The NOEC for the reproduction was determined directly from the observation data to be 0.800 mg/L.
The most sensitive effect in this study was the reduction of the mean dry body weight and mean total body length of the parent daphnids at the concentration level 0.800 mg/L. At the concentration level 0.320 mg/L no effects were observed. The EC10- and the EC50-values for the adult mortality was calculated by sigmoidal dose-response regression.

NOECReproduction : 0.800 mg/L

EC10, Adult Mortality : 0.712 mg/L
EC50, Adult Mortality : 0.974 mg/L (CI: 0.819 - 1.16 mg/L)

The recoveries in the fresh media were in the range of 49 to 121 % of the nominal values. In the old media (after 48 h or 72 h) the recoveries decreased to values in the range of 25 to 86 %. These analysed samples contained the mobile fraction of the test item as well as that adsorbed onto glassware. Biodegradation as possible reason for this is very unlikely considering the short time frame between the refreshments of the test solutions. The adsorbed fraction of the test item concentration was determined exemplarily at the concentration level 0.800 mg/L to be 0.189 mg/L, corresponding to 24 % of the nominal concentration. Adsorption to the glassware was limited and low. The limited concentration decrease between fresh and old media is most likely caused by thermodynamically more favourable redistribution of the sorbed fraction resulting in an additional sorption to suspended matter and DOC. 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 are given 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 Tallowtripropylenetetramine (CAS no. 68911-79-5, batch number S001255) was conducted according to OECD 211 (2008) from 2009-07-22 to 2010-01-18, with the definitive exposure phase between 2009-07-22 to 2009-08-12  at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str. 1, D-31157 , Germany .

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.

Tallowtripropylenetetramine (CAS No. 68911-79-5) 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 alkyl polyamines 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 polyamines 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 polyamines 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 Tallowtripropylenetetramine 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 Tallowtripropylenetetramine was analytically verified in the concentration levels 0.320, 0.800 and 2.00 mg/L and in the control via LC-MS/MS of samples taken on days 0, 7, 9, 14 (fresh media) and on days 2, 9, 16 (old media, 48 h) and 12 (old media, 72 h). Due to the mortality of all parent animals in concentration level 2.00 mg/L on day 3, the concentration level 0.800 mg/L was analytically verified instead at the remaining sampling. The freshly prepared stock solutions of 10 mg test item/L were analytically verified too and gave recoveries in the range of 86 to 133 % of the nominal value.
Recoveries between 85 to 115 % were obtained in the fresh media for the test groups exposed to 0.800 and 2.00 mg/L. In the fresh media recovery rates between 49 to 121 % were obtained in the test group exposed to 0.320 mg test item/L.

In the old media recoveries decreased to values in the range of 25 to 86 % of the nominal values. The old media samples were analysed including the sorbed fraction to glassware. In general lower recovery rates were observed for the samples at the end of every batch independent of the exposure concentration. Biodegradation as possible reason for the observed decrease of the test concentration during the exposure period is very unlikely considering the short time frame between the refreshments of the test solutions. The sorption of the test item to glassware was determined exemplarily at the concentration level 0.800 mg/L. The concentration observed was 0.189 mg/L, which corresponds to 24 % of the nominal test concentration. The observed concentration decrease between fresh and old media is thus only for a small fraction caused by sorption to glassware. The remaining reduction is most likely caused by thermodynamically more favourable redistribution of the sorbed fraction resulting in an additional sorption to suspended matter and DOC. 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 are given based on the nominal test item concentrations.

·   The average number of juveniles per parent in the control group was 144 after 21 days. The reproductive output was not statistically significant increased or reduced in any of the tested concentration levels when compared to the control (One Way Analysis of Variance, p = 0.05). At the concentration levels 2.00 and 5.00 mg/L no offspring was produced due to mortality of all parental daphnids before day 3 of the exposure phase. 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.

·   The coefficient of variation of the mean number of living offspring produced per parent alive in the control group was 5 %.

·   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 statistical significance determined at any of the tested concentration levels.

No stillborn juveniles and aborted eggs were produced by the control group. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was below 1 % at all test groups, which is not biologically significant.

·   The mean day of release of the first brood was day 8.0 in the control group. At the tested concentration levels 0.128 to 0.800 mg/L the mean day of release of the first brood was in the range of 7.7 to 8.1. At the concentration levels 2.00 and 5.00 mg/L no daphnid survived until the end of the test. The first brood was released until day 9 by all surviving daphnids of the control group and the tested concentration levels. Five broods were released by all surviving animals of the control and the test groups.

In this study the test item did not induce statistically significant effects on reproduction. A summary of all test item related effects on reproduction is given in Table 1. 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.

Table 1:        Test Item Related Effects on Reproduction (NOEC 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)	144 ± 7	145 ± 15	160 ± 9	144 ± 20	--	--
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent	5	11	6	14
Mean Intrinsic Rates of Natural Increase	0.50	0.53	0.53	0.49	--	--
Appearance of First Brood (Mean Day)	8.0	7.7	7.8	8.1	--	--
Number of Broods	5	5	5	5	--	--
NOEC	0.800 mg/L
EC50, Reproduction with Confidence Interval (CI) p = 95 %	Not determinable (CI: Not applicable)

SD    = Standard deviation

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

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

·   The test item induced significant adult mortality of 100 % at the tested concentration levels 2.00 and 5.00 mg/L after 21 days. At the other concentration levels and the control no significant mortality (≤ 20 %) of parent animals was observed. The EC₁₀ for adult mortality after 21 days was calculated to be 0.712 mg/L. The EC₅₀for adult mortality after 21 days was calculated to be 0.974 mg/L (CI: 0.819 - 1.16 mg/L).

·   The mean dry body weight of the parent daphnids was biologically significantly reduced to 0.94 mg per daphnid at the tested concentration level 0.800 mg/L when compared to the control (reduction: 32 %). The mean dry body weight at the concentration levels 0.128 and 0.320 mg/L were determined to be 1.26 and 1.28 mg per daphnid, which is comparable to the control group with a mean dry body weight of 1.39 mg per daphnid.
The mean value of the total body lengthin the concentration levels 0.128 and 0.320 mg/L were determined to be 5.67 and 5.81 mm per daphnid and 5.86 mm per daphnid in the control group. At the concentration level 0.800 mg/L the mean total body length was statistically significantly reduced to 5.28 mm per daphnid. The total body length of the concentration levels 0.128 to 0.800 mg/L were compared to the control using Kruskal-Wallis One Way Analysis of Variance on Ranks (p = 0.05), because normality test failed.

The most sensitive effect in this study was the reduction of the mean dry body weight and mean total body length of the parent daphnids at the concentration level 0.800 mg/L. At the concentration levels ≤ 0.320 mg/L no effects were observed.

Table 2:    Test Item Related Effects on Adult Mortality (EC₁₀ and 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 [%]	10	10	10	20	100^1)	100^1)
Parent Animals: Mean Dry Weight [mg]	1.39	1.26	1.28	0.94^2)	--	--
Parent Animals: Mean Body Length [mm]	5.86	5.61	5.81	5.28^3)	--	--
EC10, Adult Mortality	0.712 mg/L
EC50, Adult Mortality with Confidence Interval (CI) p = 95 %	0.974 mg/L (CI: 0.819 - 1.16 mg/L)

¹⁾       = Mortality > 20 % biologically significant

²⁾       = Biologically significant

³⁾       = Statistically significant (Kruskal-WallisOne Way Analysis of Variance on Ranks, p = 0.05)

·       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 1.29 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 20