Tall oil, reaction products with diethylenetriamine

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

It is considered appropriate to address the data requirements for DTO_DETA by read-across to the available studies on the main components of DTO_DETA: AAI_DETA and Rosin.
DTO_DETA and AAI_DETA are each a mixture of constituents which include monoamide, diamide, residual amine and imidazoline (mono-, di- and tri-condensate) chemical structures. The substances therefore have common functional groups based on amide, amine and imidazoline moieties and are sufficiently similar in terms of chemical structure to support a read-across approach.
DTO_DETA contains comparatively lower levels of imidazolines and higher levels of resin acids than AAI_DETA and therefore consideration of data for resin acids is also considered necessary. The main resin acid in DTO_DETA is abietic acid, but abietic acid derivatives and other acids, such as pimaric acid, are also found in notable quantities, and the resin acids collectively are known as ‘rosin’. DTO_DETA contains upto 25% unreacted rosin, and taking into account the compositional information available for the rosin in DTO_DETA and Rosin (CAS# 8050-09-07, EC# 232-475-7), the latter was considered appropriate for read-across to DTO_DETA.
The AAI_DETA dataset is worst case with regards to biodegradability and aquatic toxicity, with the dataset for Rosin showing a lower toxicity profile. Therefore, AAI_DETA is considered to be a worst case read-across source for the DTO_DETA target substance in the context of long-term toxicity to aquatic invertebrates.

Reason / purpose for cross-reference:

read-across source

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

270 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

270 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Parental

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

311 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Parental

Remarks on result:

other: (270-810)

Details on results:

- Mortality of parent animals:
The test item induced significant adult mortality at the concentration level 810 µg/L (100 %) after 21 days. At the other concentration levels and in the control no biologically significant mortality (≤ 20 %) of parent animals was observed. The EC50 for adult mortality after 21 days was calculated to be 311 µg/L (confidence limits: 270- 810 µg/L).
- No. of offspring produced per day per female:
Four to five broods were released by all surviving animals of the control and concentration levels 10.0 to 270 µg/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 270 µg/L when compared to the control (One Way Analysis of Variance, DUNNETT´S method p = 0.05). The reproductive outputs at the concentration levels 10.0 to 90.0 µg/L was comparable to the control group
The coefficient of variation of the mean number of living offspring produced per parent alive at the control group was calculated to be 11 %. At the concentration levels 10.0 to 270 µg/L the calculated coefficient of variation was in a range comparable to the control and is considered to be not biologically significant (coefficients of variation < 25 %).
- Body length and weight of parent animals:
The mean dry body weights of the surviving parental daphnids, determined at the end of the study, at the concentration levels 10.0 to 270 µg/L were in the range of 0.92 to 0.99 mg and comparable to the mean value of 0.91 mm per daphnid at the control group. The mean values of the total body length of the surviving parental daphnids at the concentration levels 10.0 to 270 µg/L were in the range of 5.58 to 5.63 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 tested concentration levels 10.0 to 270 µg/L and the control groups producing juveniles was between day 7 and day 8

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 was carried out using One Way Analysis of Variance, DUNETT´S method (p = 0.05) and the statistical evaluation of the intrinsic rates of natural increase 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 estimation of the EC50-value for adult mortality and the reference item was carried out by using sigmoidal dose-response regression. The confidence intervals for the EC50-value 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 EC50-value of adult mortality the highest concentration causing no effect and the lowest concentration producing 100 % were 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 alive in the Control and Test Groups after 21 Days

Nominal conc. [µg/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		[%]	[%]
810	--	--	--	--	--	--	--	--	--	--	--	0	n.a.		n.a.	n.a.	n.a.
270	135	135	133	141	176	148	132	--	126	143	1269	9	141   ±	15	10	-16.5	yes
90.0	131	135	111	128	124	102	91	121	109	133	1185	10	119   ±	15	12	1.65	no
30.0	108	114	142	118	141	116	145	111	126	126	1247	10	125   ±	14	11	-3.31	no
10.0	134	125	92	133	111	118	131	126	134	125	1229	10	123   ±	13	11	-1.65	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
[µg/L]	1	2	3	4	5	6	7	8	9	10	Mean Day
810	--	--	--	--	--	--	--	--	--	--	--
270	8	7	8	8	7	8	7	--	7	8	7.44
90.0	7	8	7	8	7	8	10	7	8	8	7.80
30.0	8	8	8	8	8	8	8	8	8	8	8.00
0.10	8	8	10	8	7	8	8	9	8	8	8.20
Control	8	8	8	8	8	8	8	8	8	8	8.00

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	0	0	10
90.0	0	0	0
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	--	--	--	--	--	--	--	--	--	--	--	--	--	--
270	5.75	5.75	5.75	6.00	5.50	5.75	5.25	--	5.00	5.50	5.58	9	8.4	0.93
90.0	5.50	5.75	5.25	5.75	6.00	5.25	5.25	5.75	5.75	5.50	5.58	10	9.9	0.99
30.0	5.75	5.50	5.75	5.50	6.00	5.50	5.25	5.75	5.50	5.75	5.63	10	9.2	0.92
10.0	5.50	5.25	5.75	5.75	5.75	5.50	5.75	5.50	5.75	5.75	5.63	10	9.5	0.95
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 long-term toxicity of the target substance DTO_DETA to Daphnia magna is estimated to be a 21 d EC10 = 270 µg/L (based on nominal test substance concentration), based on read-across from a study testing AAI_DETA. Corrected for active ingredient, which is 94.4%, the EC10 is reduced to 254.9 µg/L.

Executive summary:

The long-term toxicity of the target substance DTO_DETA to Daphnia magna is estimated to be a 21 d EC₁₀ = 270 µg/L (based on nominal test substance concentration), based on read-across from a study testing AAI_DETA. Corrected for active ingredient, which is 94.4%, the EC₁₀ is reduced to 254.9 µg/L.

As explained in the justification for type of information, DTO_DETA and AAI_DETA are each a mixture of constituents which include monoamide, diamide, residual amine and imidazoline (mono-, di- and tri-condensate) chemical structures. The substances therefore have common functional groups based on amide, amine and imidazoline moieties and are sufficiently similar in terms of chemical structure to support a read-across approach.

DTO_DETA contains comparatively lower levels of imidazolines and higher levels of resin acids than AAI_DETA and therefore consideration of data for resin acids is also considered necessary. The main resin acid in DTO_DETA is abietic acid, but abietic acid derivatives and other acids, such as pimaric acid, are also found in notable quantities, and the resin acids collectively are known as ‘rosin’. DTO_DETA contains upto 25% unreacted rosin, and taking into account the compositional information available for the rosin in DTO_DETA and Rosin (CAS# 8050-09-07, EC# 232-475-7), the latter was considered appropriate for read-across to DTO_DETA.

The AAI_DETA dataset is worst case with regards to biodegradability and aquatic acute toxicity, with the acute dataset for Rosin showing a lower toxicity profile. No long term data is available for Rosin. Therefore, AAI_DETA is considered to be a worst case read-across source for the DTO_DETA target substance in the context of long-term toxicity to aquatic invertebrates.

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 reduction in the concentration of about 70% is observed. 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 hydrolyse 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 assessment as no significant adsorption to glassware was observed and the organisms were thus fully exposed to the nominal concentration based on active ingredient.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-02-22 to 2010-08-11

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

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

Specific details on test material used for the study:

- Name of test material (as cited in study report): Tall oil diethylenetriamine imidazoline (CAS No. 68442-97-7)
- Physical state: Liquid, amber, viscous
- Analytical purity: 94 %
- Purity test date: 2009-05-11
- Lot/batch No.: S000922
- Expiration date of the lot/batch: 2017-07-02
- Water solubility: Insoluble
- Density: 980 kg/m3 at 20 °C
- pH: 10 - 12 at 75 % solution
- Stability under test conditions: Not specified
- Storage condition of test material: Room temperature, protected from moisture and light, under nitrogen

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 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 30.0 µg/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: 10.0 - 30.0 - 90.0 - 270 - 810 µg/L
- Controls: 10 replicates of diulution 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
[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
810 114 119 n.a. n.a. n.a. n.a.
270 -- -- 127 120 116 118
Control 100 100 76 116 112 112

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
810 21.9 21.1 n.a. n.a. n.a. n.a.
270 -- -- 21.2 21.7 19.4 21.4
Control 19.1 21.5
19.0 21.7 19.0 21.9

pH:

pH-Values
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
810 7.62 7.55 n.a. n.a. n.a. n.a.
270 -- -- 7.84 7.60 7.97 7.55
Control 8.07 7.79 8.07 7.70 8.11 7.59

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 18 Day 21
Feb. 22nd Feb. 24th March 1st March 3rd March 12th March 15th
810 8.57 5.71 n.a. n.a. n.a. n.a.
270 -- -- 8.85 7.60 9.39 7.51
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 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.

Location D-31171 Nordstemmen
Sampling Date 2009-12-15

Weather conditions
on Day of Sampling Cloudy, ca -1 °C
Colour Yellowish, clear
pH 7.97
Conductivity [µS/cm] 386
Dissolved Oxygen [mg O2] 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: 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 50 20 35 100 100 100
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.:

270 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

270 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Parental

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

311 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

parental

Remarks on result:

other: (270-810)

Details on results:

- Mortality of parent animals:
The test item induced significant adult mortality at the concentration level 810 µg/L (100 %) after 21 days. At the other concentration levels and in the control no biologically significant mortality (≤ 20 %) of parent animals was observed. The EC50 for adult mortality after 21 days was calculated to be 311 µg/L (confidence limits: 270- 810 µg/L).
- No. of offspring produced per day per female:
Four to five broods were released by all surviving animals of the control and concentration levels 10.0 to 270 µg/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 270 µg/L when compared to the control (One Way Analysis of Variance, DUNNETT´S method p = 0.05). The reproductive outputs at the concentration levels 10.0 to 90.0 µg/L was comparable to the control group
The coefficient of variation of the mean number of living offspring produced per parent alive at the control group was calculated to be 11 %. At the concentration levels 10.0 to 270 µg/L the calculated coefficient of variation was in a range comparable to the control and is considered to be not biologically significant (coefficients of variation < 25 %).
- Body length and weight of parent animals:
The mean dry body weights of the surviving parental daphnids, determined at the end of the study, at the concentration levels 10.0 to 270 µg/L were in the range of 0.92 to 0.99 mg and comparable to the mean value of 0.91 mm per daphnid at the control group. The mean values of the total body length of the surviving parental daphnids at the concentration levels 10.0 to 270 µg/L were in the range of 5.58 to 5.63 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 tested concentration levels 10.0 to 270 µg/L and the control groups producing juveniles was between day 7 and day 8

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 was carried out using One Way Analysis of Variance, DUNETT´S method (p = 0.05) and the statistical evaluation of the intrinsic rates of natural increase 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 estimation of the EC50-value for adult mortality and the reference item was carried out by using sigmoidal dose-response regression. The confidence intervals for the EC50-value 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 EC50-value of adult mortality the highest concentration causing no effect and the lowest concentration producing 100 % were 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 alive in the Control and Test Groups after 21 Days

Nominal conc. [µg/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		[%]	[%]
810	--	--	--	--	--	--	--	--	--	--	--	0	n.a.		n.a.	n.a.	n.a.
270	135	135	133	141	176	148	132	--	126	143	1269	9	141   ±	15	10	-16.5	yes
90.0	131	135	111	128	124	102	91	121	109	133	1185	10	119   ±	15	12	1.65	no
30.0	108	114	142	118	141	116	145	111	126	126	1247	10	125   ±	14	11	-3.31	no
10.0	134	125	92	133	111	118	131	126	134	125	1229	10	123   ±	13	11	-1.65	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
[µg/L]	1	2	3	4	5	6	7	8	9	10	Mean Day
810	--	--	--	--	--	--	--	--	--	--	--
270	8	7	8	8	7	8	7	--	7	8	7.44
90.0	7	8	7	8	7	8	10	7	8	8	7.80
30.0	8	8	8	8	8	8	8	8	8	8	8.00
0.10	8	8	10	8	7	8	8	9	8	8	8.20
Control	8	8	8	8	8	8	8	8	8	8	8.00

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	0	0	10
90.0	0	0	0
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	--	--	--	--	--	--	--	--	--	--	--	--	--	--
270	5.75	5.75	5.75	6.00	5.50	5.75	5.25	--	5.00	5.50	5.58	9	8.4	0.93
90.0	5.50	5.75	5.25	5.75	6.00	5.25	5.25	5.75	5.75	5.50	5.58	10	9.9	0.99
30.0	5.75	5.50	5.75	5.50	6.00	5.50	5.25	5.75	5.50	5.75	5.63	10	9.2	0.92
10.0	5.50	5.25	5.75	5.75	5.75	5.50	5.75	5.50	5.75	5.75	5.63	10	9.5	0.95
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 level 810 µg/L was the most sensitive effect in this study. In this study no reduction of reproductive output, but hormesis was observed. The reproductive output was statistically significantly increased at 270 µg/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: 270 µg/L

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

EC10, Adult Mortality : 270 µg/L
EC50, Adult Mortality : 311 µg/L (confidence limits: 270 - 810 µg/L)
NOECAdult Mortality : 270 µg/L


The recoveries in the fresh media were in the range of 73 to 86 % of the nominal values. In the old media (after 48 h or 72 h) the recoveries decreased to values in the range of 38 to 60 %. 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 30 µg/L on day 21 to be < LOD, which corresponds to < 0.0833 % 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. 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 diethylenetriamine imidazoline (CAS no. 68442-97-7, batch number S000922) was conducted according to OECD 211 (2008) from 2010-02-22 to 2010-08-11 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 diethylenetriamine imidazoline (CAS No. 68442-97-7) 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 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 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 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 diethylenetriamine imidazoline 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 Tall oil diethylenetriamine imidazoline 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, 18 (fresh media) and on days 2, 9 (old media, 48 h) and 21 (old media, 72 h).
The recoveries in the fresh media were in the range of 73 to 86 % of the nominal values. In the old media (after 48 h or 72 h) the recoveries decreased to values in the range of 38 to 60 %. 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 30 µg/L on day 21 to be LOD, which corresponds to 0.0833 % 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. 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 270 mg/L when compared to the control (One Way Analysis of Variance, Dunnett´s method 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 reproduction output, but hormesis was observed. An EC₁₀- or EC₅₀-value for the reproductive output could not be calculated, because no effects ≥ 10 % (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 in the range of 0.49 to 0.51 and 0.49 at the control. For details see Table 1, Table 5 and Figure 2. The IR of the surviving daphnids of the treatment groups were compared to the control by One Way Analysis of Variance (Dunnette´s method p = 0.05). There is no statistical significant difference determined at the concentration levels. At the concentration level of 810 µg/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 ≤ 1 % at the tested concentration levels. At the control no dead juveniles or aborted eggs were observed.

Effects concerning the reproduction were not observed in this study. A summary of all test item related effects on reproduction is presented in Table 1.

Table 1:        Test Item Related Effects on Reproduction and EC_{50, Reproduction}

Effects	Nominal Concentration [µg/L]
	Control	10.0	30.0	90.0	270	810
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)	121 ± 14	123 ± 13	125 ± 14	119 ± 15	141 ± 15^1)	--
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent	11	11	11	12	10	--
Number of Dead Juveniles and Aborted Eggs	0	12	12	12	8	--
Mean Intrinsic Rates of Natural Increase	0.49	0.49	0.50	0.49	0.51	--
Appearance of First Brood [Mean Day]	8.0	8.2	8.0	7.8	7.4	--
Mean Number of broods	5.0	4.9	5.0	4.9	5.0	--
NOEC (adverse effect)	270 µg/L
EC50, Reproduction with Confidence Interval (CI) p = 95 %	Not determinable (CI: Not applicable)

A summary of test item related effects concerning the adult mortality and growth (weight, length) is presented in Table 2. The adult mortality at the concentration level 810 µg/L was the most sensitive effect in this study.

·   The test item induced significant adult mortality at the concentration level 810 µg/L (100 %) after 21 days. At the other concentration levels and in the control no significant mortality of parent animals was observed. The EC₅₀ for adult mortality after 21 days was calculated to be 311 µg/L (confidence limits: 270- 810 µg/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 10.0 to 270 µg/L were in the range of 0.92 to 0.99 mg and comparable to the mean value of 0.91 mg per daphnid at the control group.
The mean values of the total body lengthof the surviving parental daphnids at the concentration levels 10.0 to 270 µg/L were in the range of 5.58 to 5.63 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 [µg/L]
	Control	10.0	30.0	90.0	270	810
Adult Mortality after 21 Days [%]	0	0	0	0	10	100^1)
Parent Animals: Mean Dry Weight [mg]	0.91	0.95	0.92	0.99	0.93	--
Parent Animals: Mean Body Length [mm]	5.55	5.63	5.63	5.58	5.58	--
EC10, Adult Mortality	270 µg/L
EC50, Adult Mortality with Confidence Interval (CI) p = 95 %	311 µg/L (CI: 270 – 810 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 1.44 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

It is considered appropriate to address the data requirements for DTO_DETA by read-across to the available studies on the main components of DTO_DETA: AAI_DETA and Rosin.

The long-term toxicity of AAI_DETA to Daphnia magna was determined as a 21 d EC₁₀ = 270 µg/L (based on nominal test substance concentration). Corrected for active ingredient, which is 94.4%, the EC₁₀ is reduced to 254.9 µg/L. No long term data is available for Rosin, but the AAI_DETA dataset is considered worst case with regards to biodegradability and aquatic acute toxicity, with the acute dataset for Rosin showing a lower toxicity profile.

The long-term toxicity of DTO_DETA to Daphnia magna is therefore estimated to be a 21 d EC₁₀ of 254.9 µg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

254.9 µg/L