Amides, tall-oil fatty, N-[3-(dimethylamino)propyl]

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 days

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

modifications related to the properties of the test substance

Principles of method if other than guideline:

Some modifications:
- Natural surface water was used for testing. Natural water contains Total Suspended Solids (TSS) and Dissolved Organic Carbon (DOC). Natural water was chosen to allow the bulk approach to be applied allowing a more environmentally realistic test to be conducted.
- Due to the low nutrient content of the chosen testing water, it was supplemented with standard OECD recommended M4 nutrients.
- The bulk approach was applied to the test methodology allowing the use of total aquatic concentrations for calculation of endpoints.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples of all the test concentrations and the stock were taken on days 0, 2, 5, 7, 9, 12, 14, 16, 19, on preparation (fresh) and on day 5 14 and after changing (used) in the 0.5 mg/L replicate. The volume of the samples of the new test concentrations was 0.5 mL which was later increased to 10 mL
after day 7. The increased sample volume, accounted better for loss of the test substance to suspended matter. Both samples were diluted with an
equal volume of leaching solution to aid stability.
In addition, extraction of the test chemical from glass wear at 0.15 mg/L was attempted.
Analysis was undertaken immediately on all samples to quantify the concentration of the test.

Vehicle:

no

Details on test solutions:

To prepare the stock solutions for every water renewal, on average 0.0049 g of test substance was weighed on an analytical balance, and then was added directly in approximately 80 mL of the test media. The obtained preparation was warmed to approximately 38 ºC whilst stirring. The stock was left an additional 30 minutes to stir and did not exceed 50 ºC during this period. The stock was then left to cool whilst stirring. After cooling, the stock was sonicated for 1 minute and the stock volume was filled up to 100 mL in a volumetric flask with test media. An opaque but homogeneous stock was achieved.
A fresh stock solution was prepared for each solution change. The pH of each stock solution was checked and found to be consistently 8.5 and was therefore not adjusted.
Test solutions were prepared by further dilution of the stock solution with the test medium. A geometric series of concentrations were used. The ratio between two consecutive concentrations was 3.2. Test vessels were filled directly from the intermediate stocks after a short stirring period. The solutions were renewed at least three times a week during the test. One control containing only test medium was included in the test.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test animals were taken from a Daphnia magna clone 5 stock, (Origin: Dr .U. Noack-Laboratorien Käthe-Paulus- Str. 1, D-31157 Sarstedt, Germany), cultured in conformity with the relevant SOP. The animals used in the test were less than 24 hours old and were obtained from parent animals reproducing parthenogenetically and aged between 2-4 weeks having previously produced at least one brood before use. Offspring from the Daphnia culture were reference tested with potassium dichromate.

At the start of the test the daphnids were randomly distributed to the test vessels. For each test concentration and control, ten vessels, with one daphnid per vessel were tested. The test duration was 21 days.
During the test, test animals were fed a diet of 0.1 - 0.2 mg of carbon per daphnid per day, in the form of the algal strain Chlorella vulgaris. The strain is continually cultured in the ECRA Environmental Chemistry laboratory and total organic carbon content has previously been measured and logged in the GLP system.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

All adults were inspected under magnification for abnormalities. Individuals were measured and dry weight was determined where possible.

Hardness:

14.3 to 16.3 °dH for controls
14.3 to 16.5 °dH for the concentration 0.5 mg/L

Test temperature:

21.0 to 21.8°C for controls
21.1 to 21.8°C for the concentration 0.5 mg/L

pH:

8.1 to 9.0 for controls
8.2 to 9.0 for the concentration 0.5 mg/L

Dissolved oxygen:

9.2 to 11.3 for controls
9.4 to 10.8 for the concentration 0.5 mg/L

Nominal and measured concentrations:

The nominal concentrations used in the study were as follows: 0.004, 0.015, 0.048, 0.15 and 0.5 mg/L. See analytical recovery table for percentage of nominal values measured.

Details on test conditions:

Test vessels: 50 mL (nominal) glass beakers were used, containing approximately 50 mL of test solution and covered by glass plates during the test.
Test room, temperature control and light regime: The test was carried out in a temperature-controlled room. The light regime was 16 h of ambient light per day, provided by fluorescent tubes.
Apparatus: The dissolved oxygen concentrations and conductivity were determined electrochemically using an oxygen electrode / conductivity electrode and meter. The pH was determined with a pH meter. The temperature was measured with a temperature sensor and recorder. Standard natural surface water parameters, ammonium and total phosphate and total hardness were measured using Dr Lange test kits.

Test medium: The test medium was prepared by mixing natural surface water with standard Elendt M4 media nutrients. A 50 % concentration of all M4 stocks diluted in natural surface water was shown to provide sufficient nutrients to support growth and reproduction of the test animals.
Natural surface water was used in this test in keeping with the bulk approach. Natural surface water was sampled from Heveadorp in the district of Renkum coordinates: 51º 58’N 5º48’E. Natural surface water was frozen until use to preserve organic contents and reduce microbial activity.

Reference substance (positive control):

yes

Remarks:

Daphnia culture referenced routinely with potassium dichromate.

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.048 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.07 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Based on the observed results the NOEC for this substance based on reproduction and adult length is 0.048 mg/L. The LOEC for this substance also based on reproduction and length is 0.15 mg/L. The Ec10 based on reproduction was calculated as 0.07 mg/L confidence limits could not be calculated. The Ec10 based on length was calculated as 0.14 mg/L confidence limits could not be calculated. The dry weight endpoint was considered unreliable due to the inaccuracy involved in weighing the low number of surviving daphnids. The adult mortality endpoint was not considered reliable due to physical effects being the most likely cause of the effects.

Any other biological effects observed
The first brood was released on day 9 in the control. No adverse effects were noted in the control replicates. All test concentrations displayed similar biological effects to a similar degree of severity with the exception of the highest 2 concentrations. Daphnids pale in colour and smaller compared to the control were noticed in all test concentrations to a similar extent. These individuals did not survive until the end of the test and appeared to be physically restricted by the test substance and/ or algae cells sticking to their bodies and antenna. This resulted in reduced movement and hence reduced feeding. During solution refreshments attempts were made to gently remove the obstructions from the antennae of the daphnids in
the test concentrations. This was successful in removing the obstructions and remobilizing the affected daphnids for a short time only. Egg development in the test concentrations only occurred in the animals that survived until the end of the test and hence overcame the physical challenge described. In most cases these animals went on to reproduce at a similar or lower level than the control a clear effect on reproduction was therefore observed.

Results with reference substance (positive control):

Offspring were reference twice a year with potassium dichromate.

Reported statistics and error estimates:

The data on reproduction was tested for normality using Shapiro-Wilk’s test and found to be normally distributed. Homogeneity of variance was confirmed by the Bartlett’s test. Analysis of variance using the Dunetts test was performed on the number of living neonates per parent alive at the end of the test.

Natural surface water parameters

Parameter	Comment
Location	Heveadorp
Sample Date	18 -11 -2009
Conditions when sampled	windy +/- 15ºc
Colour	clear-light yellow
Conductivity	331 µs/cm
NPOC	3.5 mg/l
TSS	17.2 mg/L
Ammonium	0.041 mg/L
Nitrate	1.74 mg/L
Total phosphate	0.163 mg/L
Total hardness	<1°dH
pH(with added nutrients)	7.8

The test solutions 0.048, 0.15, and 0.5 mg/L and the stock solutions were all found to be in the region of ±20% of the nominal concentrations. The lower concentrations proved to be less stable and measured at <80% of the nominal value or fell below the detection limit. The recoveries from the higher concentrations and the stock demonstrate that the correct amount of test material was added to the test vessels in keeping with the bulk approach. However the lower concentrations were not found at levels above 80 % most likely due to loss to suspended matter and possibly to glass. This is an inherent characteristic of the test substance and is proportionally more significant at lower concentrations I.e. (a greater percentage of the nominal values). The study director considers that the correct amount of test substance was added and nominal concentrations will be used for end point calculations in keeping with the bulk approach.

Extraction of the test chemical from glass wear at 0.15 mg/L was attempted. This method was not successful in removing the test substance from glass. An estimation of substance loss to glass was therefore not possible.

A comparative calibration curve was made in test water to investigate the behaviour and recovery of test chemical in test water in comparison to solvent based calibration curve. A leaching solution to aid substance stability was not used. A valid calibration curve in water was not possible to create and recovery in all but the highest concentration was not possible. This is likely due to relatively poor solubility and strong binding characteristics of the test chemical to suspended solids in the test media and potentially also to glass. Use of solvent-based calibration curves was therefore justified as the only means to quantify the test chemical. Rapid loss of the test chemical to the organic components of the test media and / or apparatus used was demonstrated. Maintenance of a significant biologically available or dissolved portion of the test chemical under environmental conditions is therefore highly unlikely.

Mean measured concentrations of test substance as a percentage of the nominal concentrations

Sample	Mean concentration (mg/L)	% of nominal
Control	< LOD	< LOD
0.0047 mg/L	< LOD	< LOD
0.015 mg/L	0.009	58
0.048 mg/L	0.038	80
0.15 mg/L	0.134	89
0.5 mg/L	0.437	87
Stock	42.0	85

Validity criteria fulfilled:

yes

Remarks:

see conclusions

Conclusions:

The following validity criteria were respected: mortality in the control did not exceed 20% during the test period and the average number of juveniles per parent in the control was 61.
Study was conducted reliably, as planned. Due to physical effects the endpoints calculated must be interpreted with care. Low solubility resulted in all
test concentrations except for the highest displaying similar adult mortality effects. Physical adhesion of the test substance to daphnids was visible
and caused considerable mortality in the early stages of the test. Daphnia surviving this initial challenge went on to survive and reproduce to the
same extent as the control in the 0.048mg/L replicate. Length data also supported this and a NOEC for 0.048mg/L was calculated for both endpoints.
There is good evidence to suggest that this was indeed a toxic effect that was measured consideration should be given to the physical effects
observed. Had these not been present a lower toxicity may have been calculated. Weight endpoint and adult mortality endpoints were not considered accurate due to physical effects and innacuracy of weighing the low number of surviving daphnia. Reproduction and length endpoints provided the most data and hence the most reliable statistical analysis.

Executive summary:

Test was conducted according to approved guidelines with GLP accreditation with deviations reported and justified. A certificate of analysis was present and chemical analysis in keeping with the bulk approach was conducted with a validated analytical method. Validity criteria and guideline recommendations for a valid test were met with the exception of chemical analysis at the end of the test which is not required as part of the bulk approach.

The exposure of the test organisms to the test chemical was in keeping with the bulk approach and use of nominal concentrations for endpoint calculations is justified. The data generated in this study can be considered an accurate representation of the chronic toxic effects of the test substance to Daphnia magna in the concentration range tested. Based on the observed results the EC10 for this substance based on reproduction and is 0.07 mg/L.

The adult mortality endpoint was not considered reliable due to physical effects being the most likely cause of the effects.

Description of key information

It is assessed from the methods of manufacture the starting materials and the similarity of the end product compositions that EC272-047-7 is sufficiently similar in all respects to EC800-353-8 that it is a sensible approach to use a read-across approach. A comparison of the two substances and a read-across justification can be found in section 13 of this dataset

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.07 mg/L

Additional information

Test was conducted with Amides, C18-unsatd., N-[3-(dimethylamino)propyl] according to approved guidelines with GLP accreditation with deviations reported and justified. A certificate of analysis was present and chemical analysis in keeping with the bulk approach was conducted with a validated analytical method. Validity criteria and guideline recommendations for a valid test were met with the exception of chemical analysis at the end of the test which is not required as part of the bulk approach.

The exposure of the test organisms to the test chemical was in keeping with the bulk approach and use of nominal concentrations for endpoint calculations is justified. The data generated in this study can be considered an accurate representation of the chronic toxic effects of the test substance to Daphnia magna in the concentration range tested. Based on the observed results the NOEC for this substance based on reproduction is 0.07 mg/L.

The adult mortality endpoint was not considered reliable due to physical effects being the most likely cause of the effects.