Reaction mass of Amines, N-tallow alkyltrimethylenedi-, mono(2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3-diaminopropane ditallate

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

10.01.2011-30.03.2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP, well described with sufficient substance quantification and identification data. Validity criteria met.

Qualifier:

according to guideline

Guideline:

other: OECD 225

Deviations:

no

Principles of method if other than guideline:

No deviation but

•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.

•The test concentrations were not generated by subsequent mixing of the spiked sediment with un- spiked sediment components. This has led to an uneven distribution and recovery of the test chemical. Observed both biologically by worm behaviour and analytically by extraction and subsequent
chemical analysis in preliminary testing. Each concentration was spiked separately and agitated for an extended period at elevated temperature with
the appropriate amount of the test chemical. This ensured an accurate exposure concentration and even distribution of the test chemical through all of the sediment components.

•Due to the sacrificial nature of the sediment sampling for chemical analysis 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, after equilibration and at the end of the test for chemical analysis.
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 taken
at the start and end of the test. Sediment samples were extracted using an accelerated solvent extraction and a manual extraction method. See
analytical methods below. Chemical analysis was conducted after spiking, after equilibration and at the end of the test in order to quantify the concentration of the test substance on the sediment. The analytical method used was an LC/ MS/MS method. Details of the method are shown below.

Vehicle:

no

Details on sediment and application:

OECD formulated sediment

Sediment according to OECD 225 was used. The table is presented in the other information section below.


Preparation of the stock solution
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 sonicated until a homogeneous milky white solution without precipitate was formed. Care was taken not to excessively overheat the stock
solution. After emptying of the initial stock solution (described below) the 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.

Preparation of the spiked sediment
The stock solutions were added to 400 g of wetted sediment and agitated for 24 hours at approximately 40ºC. 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 and then
transferred evenly into the test vessels and left to equilibrate for a 6 day period under gentle aeration. Identical procedures without the test chemical were followed for control sediment. The following test concentrations were prepared: 45, 90, 180, 360 and 720 mg/kg dw.

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

Test animals
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. 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 twice 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

Post exposure observation period:

Post exposure worms were counted and left to purge for 48 Hours in fresh water to allow more accurate dry weight determination. Worms were
observed for any visible abnormalities. No abnormalities were obseved except for the highest concentration being slightly smaller than in the other
concentrations and the control.

Hardness:

Hardness was measured ( as calcium carbonate) but calcium carbonate was used to stabilize sediment pH. The water hardness was therefore
influenced and was higher at the end of the study than at the beginning. There was however little variation between replicates and between the
control and the highest concentration. The hardness measurements demonstrated concistancy of conditions between replicates and the control
but did not indicate acurate hardness of the dilution water. The composition of water used for dilution in indicated below in other information.

Test temperature:

Min 19.0ºC Max 20.9ºC

pH:

pH Water Min 8.0 Max 8.5
pH Sediment Min 6.4 Max 6.5

Dissolved oxygen:

Min-7.0 mg/L Max- 9.1mg/L

Salinity:

Not Measured

Ammonia:

Min- 0.08mg/L Max 2.72 mg/L

Nominal and measured concentrations:

The results of the measured concentrations in the sediment is presented below in any other information results. The nominal test concentrations
were as follows:
45, 90, 180, 360 and 720 mg/kg dw.

Details on test conditions:

Method

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 6 days to equilibrate. Equilibration took place under the same conditions as the final test with gentle aeration to allow stabilization of the microbial component and distribution of the test chemical between the overlying water and sediment.

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 labelled and gently aerated for the full test duration. 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 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 transferred to clean continually aerated DSW for 48 hours to purge the worms of ingested
sediment. Then they were then transferred to appropriate vessels for dry weight determination. Dry weight determination was carried out by weighing of the oven dishes before use and then placing the entire worm population per replicate in the dish and oven drying for 24 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

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

86 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Analytical Recovery >80% of nominal. (Nominal concentrations used)

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

180 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Analytical recovery >80% of nominal (Nominal concentrations used)

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

237 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Dry Weight

Remarks on result:

other: Analytical recovery >80% ( based on Nominal Concentrations)

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

360 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Dry Weight

Remarks on result:

other: Analytical recovery >80% ( based on Nominal Concentrations)

Details on results:

Extraction of the sediment at the end of the test gave analytical recoveries of around or greater than 80% of the nominal concentrations. Nominal
concentrations were therefore used for calculating of the endpoints. Due to the inaccuracies involved with reproduction counts not taking into
account the size of the individual the dry weight enpoint is considered the most accurate as this is dertermined per replicate as a whole and is not
influenced by the presence of small or large worms.

Biological observations
During the test period no abnormal behaviour (e.g. sediment avoidance) in the test concentrations was observed. Worms burrowed into all
concentrations and were visibly feeding. Production of faecal pellets was observed at all concentrations in all replicates. In replicate 2 of 360
mg/kg no biological activity was observed after 31/1/2011. In this replicate an aeration malfunction occurred subsequently influencing this
replicate. This effect was not concentration (test substance) related and was therefore not used in data calculations. All worms were active at the
time of counting and no malformations were visible. Two dead worms were found at 180 mg/kg in replicates 2 and 4. Worms appeared slightly smaller and thinner at higher concentrations. Grouping of the worms at the highest concentration was also observed. At all other concentrations the distribution of the animals was even throughout the sediment.

Results with reference substance (positive control):

The laboratory culture was reference tested as indicated in :
"German Federal Environment Agency (2005) Validation of a Sediment Toxicity Test with Endobenthic Aquatic Oligochaete Lumbriculus Variegatus by an International Ring Test. FKZ 20267 429" as part of the GLP maintainance. The results obtained fell inbetween the minimum and maximum
values obtained in the ring test. The culture was therefore considered suitible for testing.

Reported statistics and error estimates:

Lumbriculus worms reproduce via a-sexual fragmentation. The number of worms observed in a test therefore depends on the timing of fragmentation and the moment of counting the worms. It is unlikely despite of the synchronisation that this fragmentation will occur exactly at the same time. The dry-weight of the total number of living worms is independent of the timing of fragmentation. It is therefore considered to be a more reliable endpoint than the reproduction based on the number of viable worms. For guideline compliance however both reproduction and dry weight endpoints were calculated using the TOXCALC version 5.023. The Dunnett`s t-test and Probit analyses were conducted to determine the NOEC/LOEC and the ECx values respectively.

Measured analytical recovery in the lowest middle and highest concentrations

Nominal Test Concentration mg/kg dw	T=28 Manual Acidified Extraction % of Nominal
Control	0
45	84.0
180	79.0
720	89.6

 

Summary of results

 

Endpoint	NOEC [mg/kg dw]	LOEC [mg/kg dw]	EC10  [mg/kg dw]	EC50   [mg/kg dw]
Reproduction	180	360	86	593
Biomass	360	720	237	638

Validity criteria fulfilled:

yes

Conclusions:

The study can be considered a reliable representation of the toxicity of the test substance to the test organism without significant restrictions.

Executive summary:

Study was conducted to GLP and to the appropriate guideline. Quantification was conducted with a validated analytical and suitible extraction methods. Sufficient test substance identification (Analytical certificate present) was also provided. Critical guideline criteria were met and minor data discrepancies, deviations and amendments were reported, discussed and excluded from valid data points where appropriate. Spiking method was adapted from the guideline without using solvent. Chemical analysis indicates spiking method was acceptable.

Description of key information

No toxicity tests with benthic organisms have been performed with "Reaction mass of Amines, N-tallow alkyltrimethylenedi-, (2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3 -diaminopropane ditallate" (R814M). The test substance is a salt of tallow-1,3 -diaminopropane (CAS no 1219010 -04 -4) and a mixture of talloil fatty acids, 2 -ethylhexoic and acetic acid.

Under aquatic environmental conditions a large extend of the salt is expected to be dissociated and ecotoxicity observed will originate mainly from tallow-1,3 -diaminopropane. No effects (NOEL>=100 mg/L) were observed for the WAF of tall oil fatty acid to algae and daphnids and for 2 -ethyl hexanoic acid the lowest NOEC (21d daphnia) was 25 mg/L (ECHA). Therefore available data on toxicity to benthic organisms by alkyl-1,3 -diaminopropanes were included in the dataset. The ecotoxicity of tallow-1,3 -diaminopropane is expected to be reduced when forming a salt with talloil fatty acids or 2 -ethylhexoic acid.

Two long-term sediment tests have been performed with hydrogenated tallow-1,3 -diaminopropane and a short term marine sediment test with oleyl-1,3 -diaminopropane.

The short term study with Corophium volutator resulted in a 10d LC50 of 433 mg/kg dw. The long term study with Lumbriculus variegatus resulted in a 28d EC10 of 86 mg/kg dw (reproduction) and No effects were observed upto 986 mg/kg dw in the 96h test with Caenorhabditis elegans (nematoda).

For the evaluation of the environmental risks of R814M these results need to be recalculated to R814M using the tallow-1,3 -diaminopropane content of 52.2% in R814M.

Recalculated Corophium volutator 10d LC50 of 829.5 mg/kg dw

Recalculated Caenorhabditis elegans 96h NOEC of 1889 mg/kg dw

Recalculated Lumbriculus variegatus 28d EC10 of 164.8 mg/kg dw (reproduction)

Key value for chemical safety assessment

EC50 or LC50 for marine water sediment:

829.5 mg/kg sediment dw

EC10, LC10 or NOEC for freshwater sediment:

164.8 mg/kg sediment dw

Additional information

No toxicity tests with benthic organisms have been performed with "Reaction mass of Amines, N-tallow alkyltrimethylenedi-, (2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3 -diaminopropane ditallate" (R814M). The test substance is a salt of tallow-1,3 -diaminopropane (CAS no 1219010 -04 -4) and a mixture of talloil fatty acids, 2 -ethylhexoic and acetic acid.

Under aquatic environmental conditions a large extend of the salt is expected to be dissociated and ecotoxicity observed will originate mainly from tallow-1,3 -diaminopropane. No effects (NOEL>=100 mg/L) were observed for the WAF of tall oil fatty acid to algae and daphnids and for 2 -ethyl hexanoic acid the lowest NOEC (21d daphnia) was 25 mg/L (ECHA). Therefore available data on toxicity to benthic organisms by alkyl-1,3 -diaminopropanes were included in the dataset. The ecotoxicity of tallow-1,3 -diaminopropane is expected to be reduced when forming a salt with talloil fatty acids or 2 -ethylhexoic acid.

Two long-term sediment tests have been performed with hydrogenated tallow-1,3 -diaminopropane and a short term marine sediment test with oleyl-1,3 -diaminopropane.

The short term study with Corophium volutator resulted in a 10d LC50 of 433 mg/kg dw. The long term study with Lumbriculus variegatus resulted in a 28d EC10 of 86 mg/kg dw (reproduction) and No effects were observed upto 986 mg/kg dw in the 96h test with Caenorhabditis elegans (nematoda).

For risk assessment the two long term results with hydrogenated tallow-1,3 -diaminopropane are used with an AF of 50 which results in a PNEC of 1.72 mg/kg dw.

This is the benthic PNEC for hydrogenated tallow-1,3 -diaminopropane and this value needs to be recalculated to R814M using the tallow-1,3 -diaminopropane content of 52.2% in R814M.

The benthic PNEC for R814M is 3.35 mg/kg dw.