Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine

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

Toxicity to sediment organisms is not a standard information requirement in Annex VIII.  

Based on the structural similarity with the diamines and the fact that the diamines are the main driver of the toxicity, read-across from the diamines category to diamines mono-oleates is considered justified.

In contrary to the aquatic tests where results of the available acute toxicity tests of N-(Oleyl alkyl)- 1,3 -propanediamine mono-oleates and one with N-(Oleyl alkyl)- 1,3 -propanediamine di-oleates are not comparable because the test solutions were prepared differently (bulk approach applied to the di-oleates), results from terrestrial and benthic tests should be comparable and should allow read across. In soil and sediment both mono- and di-oleates are expected to dissociate completely because through sorption of the cationic groups the dissociation equilibrium will lean to the side of full dissociation and results from diamines, mono and di-oleates should be identical when corrected for difference in molecular weight (MW). Based on these considerations read-across from Diamines and Diamines di-oleates to Diamines mono-oleates is considered justified

For the diamines three long-term sediment tests were performed.

Additional information

Based on the structural similarity with the diamines and the fact that the diamines are the main driver of the toxicity, read-across from the diamines category to diamines mono-oleates is considered justified.

In contrary to the aquatic tests where results of the available acute toxicity tests of N-(Oleyl alkyl)- 1,3 -propanediamine mono-oleates and one with N-(Oleyl alkyl)- 1,3 -propanediamine di-oleates are not comparable because the test solutions were prepared differently (bulk approach applied to the di-oleates), results from terrestrial and benthic tests should be comparable and should allow read across. In soil and sediment both mono- and di-oleates are expected to dissociate completely because through sorption of the cationic groups the dissociation equilibrium will lean to the side of full dissociation and results from diamines, mono and di-oleates should be identical when corrected for difference in molecular weight (MW). Based on these considerations read-across from Diamines and Diamines di-oleates to Diamines mono-oleates is justified

For diamines, four test results are available regarding the sediment toxicity. Three long-term studies and one short-term study. The long-term studies were performed with the hydrogenated diamine C16 -18 with Lumbriculus variegatus and Caenorhabditis elegans. The long-term study with Caenorhabditis elegans showed no effects upto 1000 mg/kg dw. Two long-term tests with Lumbriculus variegatus were performed applying two different spiking approaches. In the first test a solvent was used to spike the test substance on the sand fraction and in the second test the test substance was spiked onto the whole sediment in the water phase at a slightly elevated temperature. In the first test with solvent spiking, a significant difference in reproduction between the normal control and solvent control was observed. It was not clear why this difference was observed but considering to unrealism in using a solvent to spike the test substance it was decided to repeat the test using an environmentally more realistic solvent free spiking procedure of the whole sediment. For this second long-term Lumbriculus variegatus a NOEC and EC10 for reproduction was observed of resp. 180 mg/kg dw and 86 mg/kg dw. The NOEC and EC10 based on dry weight of resp 360 and 237 mg/kg dw are higher probably because the moment of splitting of the worms is slightly influenced by the test substance.

The spiking procedure using a solvent to spike the sand fraction is unrealistic for cationic surfactants. Cationic surfactants which may enter surface water are normally sorbed to dissolved organic matter or suspended matter and may redistribute slowly to thermodynamically more favourable sites when available. Quartz sand has a very low CEC and no organic matter. The use of natural sediment spiked without using solvent is far more realistic and could allow a more evenly distribution of the test substance over the sediment. In addition, it would allow the ingestion of the test substance more realistically. Finally, the solvent apparently had a positive influence on the reproduction which limits the reliability of the result.

 

PNEC sediment based on EPM

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

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

Where:        

PNECaquatic bulk                       =      6.46 µg/L

Kpsusp                                 =      60136 L/kg

SUSPwater                          =      15 mg/L (EU TGD, 2003)

 

The PNECsed is then calculated using the equations detailed in the TGD (2003):

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

Where:      

PNECaquatic dissolved          =      3.4 µg/L

Ksusp-water                          =      15035 m3/m3

RHOsusp                             =      1150 kg/m3(TGD, equ. 18)

PNECsed-EPM                     = 204 mg/kg dw without correction for additional exposure via ingestion