Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Increased levels of NaHCO3 (75 mg/L) was added to the test medium to assist with pH stabilization this differs with the guideline recommended concentration of 50 mg/L.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken in the normal manner (water samples) at the start and end of the test from all concentrations and the control replicate. Samples were diluted 1:1 with leaching solution prior to analysis.
At least 10 mL was sampled in each case. Samples containing algae were filtered using a 0.45 μm filter to remove algae prior to analysis. Filters were primed with the relevant solution before use. Further dilution to within the range of the calibration curve was then carried out as required in leaching solution and samples were then added to HPLC vials. The samples were analyzed as soon as possible after sampling no storage took place.

Vehicle:

no

Details on test solutions:

The test substance is sufficiently soluble to dilute the test concentrations from a stock solution. To prepare the stock solution 0.0102 g of test substance was weighed on an analytical balance 80 mL of test medium was added and stirred. A clear homogeneous stock resulted. The stock was then made up to 100 mL exactly with test medium. The stock was kept in motion while used for subsequent dilutions to generate the test concentrations.
The pH of the stock solution was checked and found to be 8.0 and was therefore not adjusted.

Preparation of the test solutions
The test solutions were prepared in triplicate by addition of the adequate amounts of stock solution to the test vessels. Then test medium was added up to the required final volume. Six control vessels containing only test medium were included in the test.
An additional non innoculated fourth sacrificial replicate was also made in the same manner for extra analytical purposes. An additional two non-inoculated parallels were prepared (one open one wrapped in aluminum foil) in deionized water only at 6.75 mg/L to allow the influence of light on test substance to be
established as additional information.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Source and maintenance of algae
The test was carried out with the freshwater unicellular algae Pseudokirchneriella subcapitata (CCAP 278/4) obtained from the Culture Collection of Algae and Protozoa SAMS Research Services Ltd. Dunstaffnage Marine Laboratory, Dunbeg, Argyll, Scotland. After purchasing this strain was cultured and maintained. Cultures on sloped agar tubes were stored at 4°C in the dark until required.
Exponentially growing cultures are maintained at 23 ± 2°C in a temperature-controlled illuminated orbital incubator and are re-cultured under sterile conditions weekly to keep the algae in this phase.
The sensitivity of the algae was checked by performing a growth inhibition test with a reference compound (potassium dichromate) twice a year , and is not used unless found to be between the set EC50 values of 0.25 to 2.0 mg/L as indicated in the study guideline.

Preparation of the inoculum
The initial stock culture was inoculated with P. subcapitata from a sloped agar tube and checked for purity by microscopic means. This algal stock culture (40 mL) of P. subcapitata was regularly transferred to fresh medium to act as inoculum for testing.
The extinction of an exponentially growing stock culture was measured. The cell density was determined using the calibration curve described in section 3.5. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1×10^4 cells/mL in the test medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

According to guideline

Hardness:

Natural surface water was also analyzed for hardness according to the SOP for the Dr Lange apparatus using the standard water hardness test kit LCK 327 and the manufacturer’s instructions. The hardness was measured as 6.5 ºdH

Test temperature:

The temperature varied from 22.9 to 23.75 °C during the test.

pH:

The pH measurements show a maximum increase of 1.2 pH units in the control from 8.0 to 9.2.
In the test with 2.25 mg/L the pH increase was 0.9 (ErC50 = 4.74 mg/L)

Salinity:

- fresh water

Nominal and measured concentrations:

The following final nominal test substance concentrations were prepared: 0.25, 0.75, 2.25, 6.75, and 20.25 mg/L which correspond with 0.0755, 0.227, 0.680, 2.039 and 6.116 mg a.i./L (active ingredient content 30.2%).
Observed concentrations of test substance Geometric mean of concentrations observed at the start and end of the test: 0.1, 0.48, 2.28, 6.04 and 17.73 mg/L which correspond with 0.0302, 0.145, 0.689, 1.824 and 5.354 mg a.i./L

Details on test conditions:

Test procedures
Culture medium was prepared by diluting the stock mineral salts in an appropriate vessel with filtered natural surface water. Adequate amounts of test substance stock solution were added to the test vessels. The Erlenmeyer flasks were then filled with test medium up to a total volume of 40 mL using a sterilized dispenser pipette. Then the inoculum was added to the vessel from an exponentially growing culture. All test concentrations were tested in triplicate. In addition, six replicates of the control were included. Non innoculated replicates (including the deionized water replicates) were incubated and prepared in the same fashion with the exception that the test organism was not added. The extinction of light in each Erlenmeyer flask was measured after 0, 24, 48 and 72 hours. Algal medium was used as a blank in the spectrophotometer.

Determination of cell concentrations
Cell concentrations were determined photometrically with a UV/VIS Spectrophotometer. Measurements were carried out at 436 nm in a cuvette with a light path of 4 cm. To establish the relation between extinction of light and cell density, a calibration curve was made which is checked yearly. From the relation between extinction (E) and counted cell number per mL (N) the following calibration curve was determined using linear regression: N = (E-0.0386)/4e-7
The calibration curve was used to determine the cell density of the inoculum before and during the test when needed.

Test medium
Natural surface water sampled on 1/3/2016 from the location as illustrated in Annex 5 was used for testing. Surface water was frozen directly after sampling. Prior to testing water was thawed, aerated, filtered to remove all suspended matter, and then supplemented with all of the OECD mineral medium ingredients as indicated in the test guideline. Due to the natural surface water not containing sufficient nutrients for meeting the guideline growth requirements OECD medium nutrients at 50% of the normal concentration were added to the natural surface water with the exception of NaHCO3 that was added at an increased concentration of 75 mg/L to maintain a more constant pH. A lower concentration of nutrients was used, as this was found to be sufficient to meet the guideline growth requirements.
The (Dissolved organic carbon) DOC of the natural water used for testing was measured once prior to the study and the weather conditions were noted during sampling and these were added to the raw data. In addition the hardness of the water was also determined. No other analysis was conducted on the natural water. Historic data has shown the sample point to be low in DOC, non-contaminated by agriculture and sufficient for algae growth under the conditions described.
Natural surface water was used as dilution water to allow a more environmentally realistic determination of the toxicity of the test chemical. The chelating properties of the test substance are such that effects to algae occur via micronutrient depletion and not via genuine toxicity. Using natural surface water mitigates this effect slightly and allows a more accurate indication of genuine toxicity to be determined.

Natural surface water
The DOC of the natural surface water was measured after sampling as 2.1 mg/L (non GLP) and 2.3 mg/L just prior to the test (GLP). The hardness was measured as 6.5 ºdH Total suspended solids (TSS) was not determined as the suspended matter was removed from the water. Conductivity was not measured.

Reference substance (positive control):

yes

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.74 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 30.2% active ingredient

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.43 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.888 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 30.2% active ingredient

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.268 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Details on results:

The ErC10 was determined as 0.888 mg/L and the ErC50 as 4.74 mg/L test substance. The EbC50 was found to be 0.772 mg/L test substance.

Results with reference substance (positive control):

The sensitivity of the algae was checked by performing a growth inhibition test with a reference compound (potassium dichromate) twice a year , and is not used unless found to be between the set EC50 values of 0.25 to 2.0 mg/L as indicated in the study guideline.

Reported statistics and error estimates:

Evaluation of data was carried out using Toxrat 2.10. The raw data was entered in the predetermined template that is specific for the test guideline conducted. The appropriate statistical tests were then conducted automatically by the software.

Table I: measured concentrations.

Sample		0h	72h		Geometric Mean
Control		< LOQ	< LOQ		N/C
0.25 mg/L		0.21	0.05		0.10
0.25 mg/L par.		N/M	< LOQ		N/C
0.75 mg/L		0.65	0.36		0.48
0.75 mg/L par.		N/M	0.2		N/C
2.25 mg/L		3.0	1.73		2.28
2.25 mg/L par.		N/M	1.75		N/C
6.75 mg/L		6.49	5.62		6.04
6.75 mg/L par. I		6.18	6.14		N/C
6.75 mg/L par. II		N/M	6.37		N/C
6.75 mg/L par. III (Wrapped)		N/M	6.39		N/C
20.25 mg/L		17.78	17.69		17.73
20.25 mg/L par.		N/M	17.39		N/C

Note: 72 hour measurements have been corrected for background response measured in a blank sample. Measurements in red have been modified to make geometric mean calculations possible. Due to the in test concentrations remaining relatively stable the non innoculated parallel data has not been used for endpoint calculations 

N/C= Not calculated

N/M = Not measured

Validity criteria fulfilled:

yes

Conclusions:

The test is valid as shown by:
The following quality criteria have been met in the present study:
· The cell density of the controls increased at least a factor 16 within 72 hours.
· The mean coefficient of variation for section-by-section specific growth rates in the control cultures must not exceed 35%.
· The coefficient of variation of average specific growth rates during the whole test period in the replicate control cultures should not exceed 7%.
· Analytical quality criteria were met.
· Algae strain used was of acceptable sensitivity.

Executive summary:

The toxicity of Amines, N-C8-22-alkyltrimethylenedi-, reaction products with sodium chloroacetate, sodium salts to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours According to OECD TG 201.

The NOEC and LOEC were not determined. The ErC10 was determined as 0.88 mg/L and the ErC50 as 4.74 mg/L. The EbC50 was found to be 0.772 mg/L.

All values in the report refer to the test material as received by the sponsor. No correction for active ingredient has been made.

The initial measured concentrations were all >80% of the nominal concentrations. Concentrations measured at the end of the test demonstrated that the exposure concentration was not maintained at +/- 20% of the nominal concentrations during the study. Geometric mean concentrations were therefore calculated for expression of the toxicity endpoints.

The ErC50 based on active ingredient is 1.43 mg/L

The ErC10 based on active ingredient is 0.268 mg/L

Description of key information

One algae study with Amines, N-C8 -18-alkyltrimethylenedi-, reaction products with sodium chloroacetate, sodium salts is available. The toxicity of amines, N-C8 -18-alkyltrimethylenedi-, reaction products with sodium chloroacetate, sodium salts to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours according to OECD TG 201. All validity criteria of the algae test were fulfilled. Geometric mean concentrations were calculated for expression of the toxicity endpoints as the test substance concentration decreased during the test.

The ErC50 based on active ingredient is 1.43 mg/L

The ErC10 based on active ingredient is 0.268 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

1.43 mg/L

EC10 or NOEC for freshwater algae:

0.268 mg/L

Additional information

The toxicity of Amines, N-C8 -18-alkyltrimethylenedi-, reaction products with sodium chloroacetate, sodium salts to an exponentially growing culture of P. subcapitata was determined over an exposure period of 72 hours according to OECD TG 201.

The NOEC and LOEC were not determined. The ErC10 was determined as 0.88 mg/L and the ErC50 as 4.74 mg/L. The EbC50 was found to be 0.772 mg/L.

The values in the report refer to the test material. No correction for active ingredient has been made.

The initial measured concentrations were all >80% of the nominal concentrations. Concentrations measured at the end of the test demonstrated that the exposure concentration was not maintained at +/- 20% of the nominal concentrations during the study. Geometric mean concentrations were therefore calculated for expression of the toxicity endpoints. Photodegradation was considered to be a possible reason for the decrease of the test substance concentration.

The ErC50 based on active ingredient is 1.43 mg/L

The ErC10 based on active ingredient is 0.268 mg/L