Propanol, iminobis-, N-(C16-18 and C18-unsatd. alkyl) derivs.

Sediment toxicity

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

sediment toxicity: short-term

Type of information:

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

Adequacy of study:

key study

Study period:

2012-09-13 to 2012-11-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See section 13.2 for the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: ISO 10872 (2010)

Deviations:

yes

Remarks:

M9 medium was prepared with 6 g/L Na2HPO4 * 2H2O instead of 6 g/L Na2HPO4. To terminate the test the multi dishes were heated for 20 minutes at 80°C instead of 10 minutes. To separate the nematodes the sediment was centrifuged three times for 10 minutes a

Principles of method if other than guideline:

M9 medium was prepared with 6 g/L Na2HPO4 * 2H2O instead of 6 g/L Na2HPO4.
To terminate the test the multi dishes were heated for 20 minutes at 80°C instead of 10 minutes.
To separate the nematodes the sediment was centrifuged three times for 10 minutes at 2200 rpm (= 1012 g) instead of 5 minutes at 800 g.
To prepare the food the bacteria suspension was centrifuged two times for 20 minutes at 2000 rpm (=836 g) instead of 20 minutes at 2000 g.
The two males found in the control were included in growth measurement.

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
A loamy sand (LUFA 2.2 soil) was used in the study. The soil was received from the LANDWIRTSCHAFTLICHE UNTERSUCHUNGS-
UND FORSCHUNGSANSTALT SPEYER, Obere Langgasse 40, 67346 Speyer, Germany. The soil was sampled at August 31th, 2012 and stored at greenhouse conditions at the test facility until use.
Parameter

*) data provided by LUFA Speyer
#) acc. to German DIN classification
LUFA-soil 2.2
Batch-No. SP2.23512
pH-value* 5.5 ± 0.2
Soil moisture before application [%] 11
Maximum water capacity* [g/100 g soil dry weight] 41.8 ± 3.0
Particle size distribution acc. to DIN*
Sand:
0.63 – 2.0 mm [%] 0.8 ± 0.3
0.2 – 0.63 mm [%] 43.4 ± 2.0
0.063 – 0.2 mm [%] 33.3 ± 1.8
Silt:
0.02 – 0.063 mm [%] 7.3 ± 0.9
0.006 – 0.02 mm [%] 4.5 ± 1.0
0.002 – 0.006 mm [%] 3.5 ± 0.7
Clay:
< 0.002 mm [%] 7.2 ± 1.2
Organic carbon content [%[* 1.77 ± 0.20
Cation exchange capacity [mVal/100 g]*
at pH 10.1 ± 0.5
Soil type loamy sand (IS)


- Details of spiking: About 24 hours before insertion of the test organisms, the respective test item amount for a stock solution was weighed out and dissolved in M9 medium (25 mL). Afterwards, definitive amounts of the stock solution were added the sediment. Additional M9 medium* was added to obtain the required test item concentrations and thereby a water content of 40 % to ensure sediment test conditions. Subsequently, the natural sediment was thoroughly mixed to ensure a homogenous distribution of the test item. Finally, 0.5 g wet weight sediment were filled into each test well
- Equilibration time: 1 day
- Equilibration conditions: 6 +/- 2 °C, dark
- Controls: Moistened test sediment was tested under the same conditions as the test replicates.

Test organisms (species):

Caenorhabditis elegans

Details on test organisms:

TEST ORGANISM
- Common name: Nematode
- Strain/clone: Wilde type strain N2 var Bristol
- Source: Caenorhabditis Genetics Center, Minneapolis
- Breeding conditions:Breeding is performed at the test facility at 20 +/- 2 °C in the dark on agar plates containing NGM (nematode growth medium) agar inoculated with Escherichia coli OP 50.
- Age of animals at beginning of exposure: First stage juveniles were obtained by filtering nematode solutions from culture plates through nylon nets (10 µm and subsequently 5 µm mesh size). The size of 30 representative juvenile nematodes, which were not used in the test, was determined prior to nematode insertion.
- Feeding during test
LB-medium* was inoculated with E. coli OP50 one day prior to the insertion of the test organisms. About 17 hours after inoculation the food bacteria were washed with M9 medium and concentrated in M9 medium by centrifugation. 0.5 mL of the food medium was provided once prior to the addition of the nematodes to each test replicate.
*) LB-medium: 10 g casein peptone, 5 g yeast extract, 10 g NaCl dissolved in 1000 mL demineralised water and autoclaved.



ACCLIMATION
- Acclimation period: None, test conditions = breeding conditions

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

natural sediment

Limit test:

no

Duration:

96 h

Exposure phase:

total exposure duration

Test temperature:

Room temperature: 19.0 - 21.5 °C

pH:

pH of the natural sediment: 5.5 +/- 0.2 (measured by the deliverer)

Nominal and measured concentrations:

Nominal: 10 - 32 - 100 - 320 - 1000 mg test item/kg sediment dry weight

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Nunc Polystyrol Multiwells (12 well microtiter plates)
- Sediment volume: 0.5 g sediment wet weight per replicate
- Overlying water volume: 0.5 mL food medium (see above)
- Aeration: no
- Replacement of evaporated test water, if any: No


EXPOSURE REGIME
- No. of organisms per container (treatment): 40
- No. of replicates per treatment group: 4
- No. of replicates per control / vehicle control: 4
- Type and preparation of food: LB-medium* was inoculated with E. coli OP50 one day prior to the insertion of the test organisms. About 17 hours after inoculation the food bacteria were washed with M9 medium and concentrated in M9 medium by centrifugation. 0.5 mL of the food medium was provided once prior to the addition of the nematodes to each test replicate.
*) LB-medium: 10 g casein peptone, 5 g yeast extract, 10 g NaCl dissolved in 1000 mL demineralised water and autoclaved.


CHARACTERIZATION OF SEDIMENT
- Composition: please see specifications above
At experimental starting (Day -1) the sediment was moistened with M9 medium to a water content of 40.0 %.
- Maturation of artificial substrate (if any): not applicable

OTHER TEST CONDITIONS
- Continuous dark
The pH of the natural soil was 5.5 ± 0.2 (measured by the deliverer). The initial soil moisture was 11.0 %. The room temperature ranged from 19.0 to 21.5 °C.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The recovery / mortality of the inserted nematodes was analysed by counting the adult nematodes under a microscope. The body length of each female adult nematode was determined with a microscale.
The reproduction was analysed by counting the juveniles in subsamples under a microscope in petri dishes with an optical raster. The total number of offspring per replicate was calculated by using the entire volume of extract.


VEHICLE CONTROL PERFORMED: no


TEST CONCENTRATIONS
- Range finding study
Range Finding Test (96 h): Recovery, Mortality and Reproduction
Test Item Concentration Recovery of adult nematodes Fertility of adult nematodes Reproduction
[mg test item/
kg sediment dry weight] [%] [%] [Offspring per replicate]
Control 83.3 100 190
10 95.0 100 405
100 60.0 100 193
1000 13.3 100 10

Reference substance (positive control):

yes

Remarks:

Benzyldimethylhexadecylammonium chloride

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

219 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (206-244)

Details on results:

- Mortality of test animals at end of exposure period:
After 4 days 97.5 % of the inserted nematodes were recovered in the liquid control.
In the natural test sediment a recovery of 80 % was observed in the control after 4 days of exposure. The resulting mortality was within the range of experimental variation considered acceptable for the control group. No statistically significant mortality was observed in the test item concentrations 10, 32 and 100 mg/kg sediment dry weight. However, at the test item concentrations 320 and 1000 mg/kg sediment dry weight the mortality was statistically significant increased
- No. of offspring produced:
The occurrence of males was 7.75 % in the control and 0 % at all test item concentrations. Thus, the male occurrence was below the validity criteria of a maximum of 10 % males per treatment group.
All surviving female nematodes in the control as well as in the test item concentrations 10 to 320 mg test item/kg sediment dry weight were observed to be fertile.
The reproduction per inserted female test organism in natural sediment was not statistically significant reduced compared to the control at the test item concentrations 10, 32 and 100 mg/kg sediment dry weight.
In the test item concentrations 320 and 1000 mg/kg sediment dry weight a statistically significant reduction in reproduction was observed. However, as almost no nematode survived at 1000 mg test item/kg sediment dry weight no reproduction was observed
- Growth:
The initial mean size of the introduced juvenile nematodes was 254 µm. In sediment the growth of the adult nematodes did not statistically significant differ compared to the control at the test item concentrations 10 to 100 mg test item/kg sediment dry weight. At 320 and 1000 mg test item/kg sediment dry weight the nematode growth was statistically significant reduced
- Morphological abnormalities: Not observed
- Behavioural abnormalities: Not observed

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels:

Effects Reference Item Benzyldimethylhexadecylammonium chloride
[mg/L]
Mortality 10
Fertility 5
Reproduction 5
Growth 5
LOEC
(Mortality) 10
NOEC
(Mortality) 5
LOEC
(Reproduction, Growth) 5
NOEC
(Reproduction, Growth) < 5
LC50 (CI)
(Mortality) 6.570 (5.0 – 10.0)
EC50
(Reproduction) < 5
EC50 (CI)
(Growth) 6.281 (5.0 – 10.0)*

Reported statistics and error estimates:

ANOVA was used for LOEC and NOEC calculations. Prior to running ANOVA a Normality and an Equal Variance Test were done. P-values for both Normality and Equal Variance Test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05.
LC50-value and confidence interval was calculated by sigmoidal dose-response regression. Calculation of the
confidence interval was carried out using standard procedures.

Recovery / Mortality after 4 Days of Exposure to Bis (2-hydroxyethyl) oleyl amine

Nominal test item concentration [mg test item/kg sediment dry weight]	[%] Recovered test organisms						Mortality [%]	Significance*
	Replicate				MV	SD
	1	2	3	4
Control	100	70	60	90	80.0	18.3	20.0	-
10	80	80	70	80	77.5	5.0	22.5	No
32	60	90	80	100	82.5	17.1	17.5	No
100	80	50	80	90	75.0	17.3	25.0	No
320	60	30	30	0	30.0	24.5	70.0	Yes
1000	0	0	0	0	0	0	100	Yes

Nematode Reproduction after 4 Days of Exposure to Bis (2-hydroxyethyl) oleyl amine

Nominal test item concentration [mg test item/kg sediment dry weight]	Number of offspring per inserted female test organism
	Replicate				MV	SD
	1	2	3	4			Inhibition %
Control	38.1	34.4	37.0	33.3	35.7	2.23	-
10	33.9	36.3	31.6	37.5	34.8	2.62	2.45
32	26.8	32.1	35.1	36.8	32.7	4.39	8.40
100	44.1	32.7	39.3	42.9	39.8	5.12	-11.3
320	18.5	10.7	13.1	1.2	10.9	7.23	69.5
1000	0.6	1.2	n.a.	n.a.	0.45	0.57	98.7

Nematode Growth after 4 Days of Exposure to Bis (2-hydroxyethyl) oleyl amine

Nominal test item concentration [mg test item/kg sediment dry weight]	Mean Growth [µm]
	Replicate				MV	SD	Inhibition [%]	Significance*
	1	2	3	4
Control	984	905	898	893	920	43.0	-	-
10	892	916	850	970	907	50.1	1	No
32	930	850	940	968	922	50.6	0	No
100	892	888	898	807	871	43.0	5	No
320	794	738	898		810	81.2	12	Yes
1000	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.

Validity criteria fulfilled:

yes

Conclusions:

In this study Bis (2-hydroxyethyl) oleyl amine (CAS No. 25307-17-9) did not induce statistically significant mortality or growth inhibition of C. elegans in the test item concentrations 10 – 100 mg/kg. Also, nematode reproduction was not affected at these test item concentrations. In the test item concentrations 320 and 1000 mg/kg statistically significant effects on mortality, growth and reproduction were observed after 96 hours of exposure.

Thus, the Lowest Observed Effect Concentration (LOEC) concerning all test parameters was set at 320 mg test item/kg sediment dry weight. The Overall No Observed Effect Concentration (NOEC) was determined to be 100 mg test item/kg sediment dry weight. All effect levels given are based on the nominal concentrations of Bis (2-hydroxyethyl) oleyl amine.
The EC10-value for the reproduction was determined to be 219 mg test item/kg sediment dry weight (Table 1).
The LC50-value was determined to be 167 mg test item/kg sediment dry weight with a confidential range from 107 to 263 mg test item/kg sediment dry weight.

Executive summary:

The effects of the test item Bis (2-hydroxyethyl) oleyl amine (CAS No. 25307-17-9) (batch no.S-001018) on the bacterivorous nematode Caenorhabditis elegans in a sediment system were determined at Dr.U.Noack-Laboratorien, Sarstedt, Germany, from September 13^thto November 21^st, 2012 with a definitive exposure period lasted from November 16^thto 20^th, 2012. The study was carried out according to ISO 10872 (2010). Test duration was 96 hours after insertion of the test organisms. The study was performed by spiking the test item into the sediment with the test item concentrations 10 - 32 - 100 - 320 - 1000 mg test item/kg sediment dry weight. Four replicates per control and test item concentration were set up. The sediment used in the study was natural standard LUFA 2.2 soil, moistened to sediment conditions.

After four days of exposure, Bis (2-hydroxyethyl) oleyl amine (CAS No. 25307-17-9) did neither induce statistically significant mortality nor growth inhibition of C. elegans in the test item concentrations 10 to 100 mg/kg. Also, nematode reproduction and growth were not affected at these test item concentrations. In the test item concentrations 320 and 1000 mg/kg statistically significant effects on mortality, growth and reproduction were observed after 96 hours of exposure. Nematode fertility was not affected in the surviving female nematodes in the test item concentrations 10 to 320 mg test item/kg sediment dry weight.

Overall, the No Observed Effect Concentration (NOEC) was determined to be 100 mg test item/kg sediment dry weight. All effect levels given are based on the nominal concentrations of Bis (2-hydroxyethyl) oleyl amine (CAS No. 25307-17-9). The EC₁₀-value for the reproduction was determined to be 219 mg test item/kg sediment dry weight.

The LC₅₀-value was determined to be 167 mg test item/kg sediment dry weight

Summary of all Effects and resulting LOEC, NOEC based on nominal concentrations

Effects	Bis (2-hydroxyethyl) oleyl amine (CAS No. 25307-17-9)
	[mg test item/kg sediment dry weight]
Mortality	320
Reproduction per gravid organism	320
Growth	320
LOEC (Mortality, Fertility, Reproduction, Growth)	320
NOEC (Mortality, Fertility, Reproduction, Growth)	100
EC10	219 (206 – 244)
LC50	167 (107 – 263)

Reference 2

Endpoint:

sediment toxicity: long-term

Type of information:

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

Adequacy of study:

key study

Study period:

19-10-2011 to 23-11-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See section 13.2 for the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: OECD 225 sediment -water lumbriculus toxicity test using spiked sediment

Deviations:

yes

Remarks:

A solvent was not used to spike the test chemical to the sediment. The sediment was spiked as a complete sediment with a refined and extended spiking procedure to allow complete distribution and equilibration of the test chemical in all components of the

Principles of method if other than guideline:

A solvent was not used to spike the test chemical to the sediment. The sediment was spiked as a complete sediment with a refined and extended
spiking procedure to allow complete distribution and equilibration of the test chemical in all components of the sediment without the use of a solvent as would occur in the environment.

The test concentrations were not generated by subsequent mixing of the spiked sediment with un- spiked sediment components. This can lead to an uneven distribution and recovery of the test chemical. Each concentration was spiked separately and agitated for an extended period with the
appropriate amount of the test chemical. This ensured a more accurate exposure concentration and even distribution of the test chemical through all of the sediment components as would occur in the environment.




Due to the sacrificial nature of the sediment sampling for chemical analysis and dry weight determinations 100 g of formulated sediment was used
per replicate to ensure enough sediment remains to allow adequate growth of the test organisms.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples of sediment were taken for chemical analysis prior to equilibration at (day -1) from the bulk spiked sediment at each concentration and as
close as possible to the end of the test (day 27) from the test vessels. Enough sediment was sampled to allow analysis, dry weight determination and
any required repeats of results. To ensure the consistency of the experimental replicates, samples at the end of the test were taken in at least 2
replicates in the sampled concentrations. Water samples were also taken as close as possible to the end of the test only as it was visible from the
initial sediment analysis that the water concentration was negligible and suspended clay in several concentrations would have prevented accurate
initial water measurements. Sediment samples were extracted using an accelerated solvent extraction and a manual extraction method.
Chemical analysis was conducted on all samples taken. The analytical method used was a validated LC-MS/MS method.

Vehicle:

no

Details on sediment and application:

Sediment composition is detailed in other information below (Table 1). This was according to OECD 225. Sediment was prepared by adding the listed
ingredients together. De ionized water was then added until the sediment reached a consistency suitable for spiking. The peat used in the formulated sediment had been previously conditioned and pH adjusted by slow addition of calcium carbonate over an extended period of 2 – 3 weeks. Peat was a
ir dried prior to mixing with the other components.

The desired test solutions were prepared separately per concentration. The appropriate amount of the test chemical needed to achieve the desired concentration in the sediment was weighed out in a glass beaker on an analytical balance. Then 80 – 100 mL of DSW was added and the substance was stirred with a Teflon stirrer until a homogeneous solution was formed. To ensure a homogeneous dispersion the stock was also ultrasonicaticated for a short period. Care was taken not to excessively overheat the stock solution. After emptying of the initial stock solution (described below) the above procedure was repeated with DSW only until no visible trace of the test substance remained in the beaker. This was repeated in the same manner for
all test concentrations required.

The stock solutions were added to 400 g of wetted sediment and allowed to turn slowly in a head over heels mixer for 24 hours at room temperature. After spiking the resulting sediment was checked for pH and adjusted with calcium carbonate if required. The resulting spiked sediment was then sampled for analysis then transferred evenly into the test vessels and left to equilibrate for a 24 hour period under gentle aeration. Identical procedures without the test chemical were followed for control sediment except 600 g was prepared. The following nominal test concentrations were prepared: 25, 50, 100, 200 and 400 mg/kg dw.

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

The test animals were taken from the environmental chemistry laboratories Lumbriculus Variegatus stock originating from Wageningen University. The test animals were cultured, sub-cultured and synchronised in conformity with laboratory Standard Operation Procedures. In preparation for
testing 4 weeks prior to the planned start of the test the animals were sub cultured to allow optimal growth and increase of body mass. 2 weeks prior
to testing the sub cultured organisms were synchronized and the tail ends of the worms were allowed to recover for a maximum of 14 days before
being used in the test. Test animals of a similar size were chosen from this synchronized batch for use in the test, animals were selected at random
from these synchronised animals for use in the test concentrations . All selected test animals were therefore considered to be of a similar
physiological state. A representative sample of this batch was sampled and the dry weight was determined to allow an increase in dry weight endpoint to be calculated at the end of the test if required. The test animals were reference tested at least once a year to check the condition of the culture as indicated in the test guideline.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Remarks:

OECD 225

Post exposure observation period:

Animals were observed post exposure for any visible abnormalities or differences from the control. This was recorded.

Hardness:

Hardness of dilution water was 13.6 ºdH or 243 mg/L as CaCo3

Test temperature:

The test room varied from 18.6 to 19.7ºC

pH:

The maximum pH variation of the overlying water was 7.8-8.5.

Dissolved oxygen:

The maximum variation of oxygen variation of the overlying water was 8.2-9.2 mg/L

Salinity:

DSW, Not measured

Ammonia:

The maximum variation of the Ammonia concentration was 0.044-0.412 mg/L. All test concentrations were continually aerated to avoid anaerobic
conditions.

Nominal and measured concentrations:

See table 6 in additional information for the nominal and for the measured concentrations.

Details on test conditions:

The test was performed as a 28 day static test. The number of animals used per concentration was 40. Animals were equally divided over 4 replicates of 10 animals and exposed to the test concentrations. The control contained 6 replicates of 10 animals. Before the addition of the worms, the spiked
sediment was left for 24 hours to equilibrate and fully settle. Equilibration took place under the same conditions as the final test with gentle aeration
to allow stabilization of the microbial component oxygenation of the water and distribution of the test chemical between the overlying water and se
diment.

Synchronized worms were randomly placed in the test fluids and the test vessels were placed in a random manner on the laboratory work surface. The test vessels were clearly labeled and gently aerated for the full test duration and filled as required to compensate for evaporation. During the test,
the animals were not additionally fed as the food components were included in the formulated sediment. The test was inspected at least 6 times a week, biological observations were recorded and relevant physical chemical parameters were measured according to the study plan. At the end of the test, surviving worms were gently sieved from the sediment with a 250µm sieve and counted for use in endpoint calculations.

Worms were considered dead if no active movement occured after stimulation. Due to the nature of the a sediment test any dead worms are likely to
have been decomposed during the test period and are very difficult or impossible to find in the sediment. Dead worms were recorded in the raw data
if observed. After counting the worms, living worms were observed and then transferred to clean continually aerated DSW for 48 hours to purge the
worms of ingested sediment. Then they were transferred to appropriate vessels for dry weight determination. Dry weight determination was
carried out by weighing of oven dishes before use and then placing the entire worm population per replicate in the dish and oven drying for 72
hours at approximately 100ºC to drive of all moisture. Reweighing after cooling allowed the dry biomass to be determined for use in endpoint
calculations

Reference substance (positive control):

yes

Remarks:

Periodically as part of GLP maintainance

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

84.6 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Remarks on result:

other: Confirmed by > 80 % analytical recovery of nominal concentration. Most reliable and sensitive endpoint reccomended for Risk assesment use

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

280.7 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Remarks on result:

other: Confirmed by > 80 % analytical recovery of nominal concentration

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

101.2 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Confirmed by > 80 % analytical recovery of nominal concentration

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

357 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Confirmed by > 80 % analytical recovery of nominal concentration

Details on results:

The average extraction efficiency of the extraction method used was 80 %. Recoveries of the test chemical at the end of the study after correction
for extraction efficiency remained > 80% of the nominal concentration and Nominal concentrations were therfore used for endpoint calculations. Due to the variation and therefore less reliable NOEC results the EC10 results are considered most relevant and reliable for risk assesment purposes whenconsidering the confidance intervals determined and the acceptance in the technical guidance of using an EC10 for this purpose. Use of the most
sensitive EC10 result is proposed for risk assesment use in this case Biomass.

Results with reference substance (positive control):

Results with a reference substance were performed as part of the GLP laboratory maintainance as indicated in the test guideline. The test animals usedmet the published ring test criteria

Reported statistics and error estimates:

Both reproduction and dry weight endpoints were calculated using the TOXCALCTM version 5.023. The Dunnett`s t-test, and the Wilcoxon rank sum
tests were conducted to determine the NOEC/LOEC values and and the ECx values were calculated using Probit / Logit analysis.

Chemical analyses

The results of the specific chemical analysis are summarized in the table below. The recovery is presented as the measured concentrations in mg/kg and as the percentage recovery from sediment (corrected for extraction efficiency). Measurements were made as close as was possible to the start and the end of the test. The test substance was quantified based on the C18 main component using the method as described in Annex 1. Water measurements were also made and these are displayed in Annex 1 only. They are considered of less importance as the test substance partitioned so strongly to the sediment that the water concentrations were negligible in comparison to the sediment concentrations.

Nominal and Measured Test Concentrations

Conc. (mg/Kg, dry weight) after correction (T=-1)	% of nominal after correction   (T=-1)	Conc. (mg/Kg, dry weight) after correction (T=27d)	% of nominal after correction   (T=27 d)
Control	---	---	---
24.5*	98.0*	20.9*	83.6*
53.8*	107.6*	40.7*	81.0*
93.5*	93.5*	90.5*	90.5*
192.2*	96.2*	186.0*	93.0*
420.1*	105.0*	320.5*	80.1*

*=correction for 84.2% extraction efficiency

Validity criteria fulfilled:

yes

Conclusions:

Based on the quality of reporting, the standard guidelines followed, the quality of the analytical techniques applied the study result is considered as a valid and reliable result. The results are considered adequate for the evaluation of the benthic hazard assessment.

Executive summary:

A 28 day sediment toxicity study with was conducted to determine the impact of the test substance: on the reproduction and biomass of the aquatic oligochaeteLumbriculus variegatus. Adult worms of a synchronised physiological state were exposed to a series of test substance concentrations spiked to the sediment. Formulated sediment according to OECD guideline 225 and reconstituted Dutch Standard Water were used as test media.

Spiking was performed via addition of the test chemical to wet sediment followed by a 24 hour agitation period. The spiked sediment was then equilibrated by adding the test water and allowing a further 24 hours period under test conditions with gentle aeration for settling and water oxygenation.

Ten test organisms per replicate (4 replicates) were then added to the test replicates and left for the full test period during which biological observations were made. At the end of the test, the effect of the test chemical on worm number and biomass per concentration was determined. Statistical analysis according to the appropriate guideline recommendations were conducted to determine if there was a significant difference from the control and to establish a dose response curve where possible.

All critical quality criteria were met. Slight modifications to the test guideline were explained and justified.

Analytical measurements to determine the test substance concentration in the sediment and in the water were carried out using a validated LC-MS/MS method.  

The study can be considered an accurate representation of the effects of the test substance to Lumbriculus variegatus.

Description of key information

No sediment test data is available for tallow di(2-hydroxypropyl)amine.

PNEC for benthic organisms can be calculated applying the equilibrium partitioning method using a Kpsed or Kdsed of 4526 L/kg (or Koc of 90520 L/kg) this leads to a PNEC of 2.2 mg/kd dw including the additional correction factor of 10 for additional exposure via ingestion.

Two valid long-term test have been performed on oleyl diethanolamine and are read across to  tallow di(2-hydroxypropyl)amine. One test is performed with Lumbriculus variegatus, an endobenthic worm, and resulted in an EC10 of 84.6 mg/kg dw and one with Caenorhabditis elegans which resulted in an EC10 of 219 mg/kg dw. From these endpoints a PNEC of 1.692 mg/kg dw can be calculated applying an assessment factor of 50. As the test substance is an ionic substance the reliability of the actual test is considered to be higher than the reliability of the EPM based PNEC.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

84.6 mg/kg sediment dw

Additional information

PNEC sediment based on EPM

In the absence of measured data the PNECsed can be calculated using the equilibrium partitioning method (EPM). According to the TGD this method uses the PNECaquaticand the sediment/water partitioning coefficient as inputs. However, since the available PNECaquaticis based on the bulk concentration present in surface water a re-calculation is necessary first:

                                    PNECaquatic, dissolved = PNECaquatic bulk/ (1 + Kpsusp* SUSPwater* 10-6)

Where:

PNECaquatic bulk               =      2.8 µg/L

Kpsusp                              =      9052 L. kg-1

SUSPwater                        =      15 mg. L-1(TGD)

 

The PNECsedis then calculated using the equations detailed in the TGD:

                                 PNECsed                    =  Ksusp-water* PNECaquatic dissolved* 1000 * 1 / RHOsusp

Where:        

PNECaquatic dissolved      =     2.465 µg/L

Ksusp-water                     =     2264 m3. m-3

RHOsusp                         =     1150 kg. m-3(TGD, equ. 18)

PNECsed-EPM                 = 4.85 mg/kg ww

                                       = 22.3 mg/kg dw

 

                                    Applying the additional safety factor for exposure via ingestion: 10

PNECsed-EPM                = 0.49 mg/kg ww

                                      = 2.2 mg/kg dw

Two valid long-term test have been performed on oleyl diethanolamine and read across. One test is performed with Lumbriculus variegatus, an endobenthic worm, and resulted in an EC10 of 84.6 mg/kg dw and one with Caenorhabditis elegans which resulted in an EC10 of 219 mg/kg dw. From this endpoint a PNEC of 1.692 mg/kg dw can be calculated applying an assessment factor of 50. As the test substance is an ionic substance the reliability of the actual test is considered to be higher than the reliability of the EPM based PNEC.