Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23rd February to 15th April 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted to GLP. This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2005

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

EC Number:

269-358-5

EC Name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Cas Number:

68227-46-3

Molecular formula:

C21H35N3O4.C3H6O3

IUPAC Name:

2-hydroxypropanoic acid 2-(dimethylamino)ethyl N-[5-({[(2-ethylhexyl)oxy]carbonyl}amino)-2-methylphenyl]carbamate

Details on test material:

- Name of test material (as cited in study report): WRS-2390TX
- Physical state: Vicous liquid
- Analytical purity: 71.2% in water
- Expiration date of the lot/batch: 15th November 2004
- Storage condition of test material: The test item was stored at ambient laboratory temperature in the dark for the duration of the study.
- Notebook number: 02-181-126

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
The definitive test was conducted with nominal WRS-2390TX concentrations of 0.04, 0.09, 0.20, 0.43 and 0.94 mg/L total test item; these concentrations were equivalent to 0.03, 0.06, 0.14, 0.31 and 0.67 mg/L (corrected for 71.2% test item purity).
- Sampling method:
In the definitive test exposure phase, samples for analysis were taken from all concentrations and the controls at the start of the study and after 96 h. Upon receipt in the analytical laboratories, the samples taken at 96 h were centrifuged at 1000 rpm for 5 min to remove algal cells. The remaining supernatant from each sample was analysed.
- Sample storage conditions before analysis:
Analysis was normally performed on the same day but when this was not possible the samples were stored under appropriate conditions until analysis could take place.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
A 100 mg/L test item solution was prepared by adding a weighed amount of test item (ca 10 mg) to a 100 ml capacity volumetric flask and bringing to volume with ISO freshwater algal medium. This solution was sonicated for ca 10 min to aid dissolution. Aliquots of the 100 mg/L test item solution (0.2, 0.45, 1.0, 2.15 and 4.7 mL) were brought to volume with ISO freshwater algal medium in 500 ml volumetric flasks to prepare the 0.04, 0.09, 0.20, 0.43 and 0.94 mg/L treatment groups, respectively.
Flasks were prepared in triplicate for each treatment group, with six flasks prepared for the control. Each flask contained 100 mL of media and was inoculated with Pseudokirchneriella subcapitata. Algal cell concentrations were determined in each test flask at 24, 48, 72 and 96 h during the definitive test.
From visual observations and cell concentration determination it was determined that the 0.94 and 0.43 mg/L treatment groups had >50% inhibition of growth after 96 h exposure to WRS-2390TX. These were inoculated into fresh medium at the rate of 1:19 (exposure medium/fresh medium) and incubated for 9 days with the re-inoculated controls under similar conditions to the exposure phase.
Algal cell concentrations were determined in each test flask at 24, 48, 72 and 96 hours during the exposure phase of the definitive test and on Day 9 of the re-inoculation phase. Samples (2 mL) were removed from each flask and combined with 20 μL Lugol’s Iodine Solution. The samples were then shaken by hand, to ensure thorough mixing with the iodine, and stored at ca 4°C until determination of cell concentration. Cell concentrations were determined within 7 days after sampling using a Compound Light Microscope and Improved Neubauer Counting Chambers.
An extra flask was prepared at the 0.94 and 0.09 mg/L treatment groups. These flasks were not inoculated with algal cells. Chemical analysis of the medium in these flasks, compared with the analysis of samples from inoculated flasks, indicate if adsorption/absorption to the algal cells occurred over the exposure period.
The pH of the prepared test item stock solutions were determined at the beginning of the definitive test. In addition, the pH of every test flask was determined after the 96 h exposure phase.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Source (laboratory, culture collection): The test species was supplied from Culture Centre of Algae and Protozoa (CCAP), Cumbria, England.
- Method of cultivation: The pre-culture used for inoculation of test vessels was healthy and uninfected and was held for 1 day, rather than 3-7 days as stated in the protocol (protocol deviation), in conditions similar to those used in the test. This was initiated from a culture incubated for 3 days and which was directly cultured from a refrigerated slope, supplied by CCAP.

- Any deformed or abnormal cells observed:
No.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

9 days

Test conditions

Hardness:

nda

Test temperature:

The temperature recorded continuously during the definitive test was in the range 22.7-23.8 °C.

pH:

The pH of the prepared test item stock solutions were determined at the beginning of the definitive test. In addition, the pH of every test flask was determined after the 96 h exposure phase.
pH at 0 hrs = 7.7-7.8; pH at 96 hrs = 7.7-10.1

Dissolved oxygen:

nda

Salinity:

nda

Nominal and measured concentrations:

The definitive test was conducted with nominal WRS-2390TX concentrations of 0.04, 0.09, 0.20, 0.43 and 0.94 mg/L total test item; these concentrations were equivalent to 0.03, 0.06, 0.14, 0.31 and 0.67 mg/L (corrected for 71.2% test item purity). The geometric mean of the initial and 96 hour analytical concentrations were 0.02, 0.03, 0.11 0.27 and 0.61 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:Glass Erlenmeyer flasks (250 mL) were used as the test vessels. Each test vessel was labelled with the project number, vessel/replicate number, test concentration and study start date.
- Initial cells density: The initial cell concentration in the test cultures was ca 1 x 10ˆ4 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Detailed composition of medium:
ISO Freshwater Algal Growth Medium was prepared by addition of weighed quantities of analytical grade chemicals to de-ionised water, to give the following concentrations:
Constituent Concentration (mg/L)
NH4Cl 15
MgCl2.6H2O 12
CaCl2.2H2O 18
MgSO4 .7H2O 15
KH2PO4 1.6
FeCl3 .6H2O 0.08
Na2EDTA.2H2O 0.1
H3BO3 0.185
MnCl2.4H2O 0.415
ZnCl2 3 x 10-3
COCl2.6H2O 1.5 x 10-3
CuCl2.2H2O 1 x 10-5
NaMoO4.2H2O 7 x 10-3
NaHCO3 50


TEST MEDIUM / WATER PARAMETERS
Algal medium as described above.


OTHER TEST CONDITIONS
The cultures and test vessels were maintained within an illuminated cooled orbital shaking incubator at a temperature range of 22-26 °C. The temperature was monitored continuously in a single control vessel for the duration of the definitive test.
The algae were provided with continuous uniform illumination obtained from universal white-type fluorescent lamps.
Light intensity on the shaker table was checked at 3 different places prior to the start of the test using a Lutron LX-101 Lux meter. This ranged from 4000 to 4300 lux in line with protocol requirements.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Counting chamber


TEST CONCENTRATIONS
- Range finding study
An initial range finding test was conducted at 1.0, 10, 100 and 1000 mg/L with an untreated control. No growth was found at any test concentration and this test was repeated at the lower concentration range to try to establish a definitive test concentration range. This data is retained in the study file and is not reported.
- Test concentrations:
A second range finding test was conducted under static conditions over a 96 h period at nominal WRS-2390TX formulation concentrations of 1.0, 0.1, 0.01 and 0.001 mg/L. An untreated control was also tested.
A 1.0 mg/L test suspension was prepared by adding a weighed amount of test item (ca 2 mg) to a 2 litre capacity volumetric flask and bringing to volume with ISO freshwater algal medium. This solution was sonicated for ca 10 min to aid dissolution. Aliquots of the 1.0 mg/L solution (0.25, 2.5 and 25 mL) were placed into 250 mL capacity volumetric flasks and made up to volume with ISO freshwater algal medium to prepare the 0.001, 0.01 and 0.1 mg/L test media, respectively.
Duplicate flasks were prepared for each treatment group and control, each containing 100 mL of test solution or untreated medium, as appropriate. The flasks were then inoculated with ca 1 x 10ˆ4 cells/mL Pseudokirchneriella subcapitata.
Algal cell concentrations were determined in each test flask after 96 hours.
- Results used to determine the conditions for the definitive study:
The results of the range finding test indicate that growth was inhibited by 16.6, 5.2, 16.3 and 99.3% at 0.001, 0.01, 0.1 and 1.0 mg/L WRS-2390TX, respectively, as indicated by cell numbers when compared with control cultures.
Based on the results of the range finding test, a definitive test was conducted over 96 h (exposure phase) plus a re-inoculation phase of 9 days.
The first phase (exposure phase) was conducted with three replicate vessels at each test concentration and six replicate vessels for the untreated control. The test concentrations selected were: 0.04, 0.09, 0.20, 0.43 and 0.94 mg/L total test item, which were equivalent to nominal concentrations of 0.03, 0.06, 0.14, 0.31 and 0.67 mg/L (corrected for 71.2% test item purity).

Based on the results of the second range finding test, a definitive test was conducted over 96 h (exposure phase) and included a re-inoculation phase of 9 days. The definitive test was conducted with nominal WRS-2390TX concentrations of 0.04, 0.09, 0.20, 0.43 and 0.94 mg/L total test item; these concentrations were equivalent to 0.03, 0.06, 0.14, 0.31 and 0.67 mg/L (corrected for 71.2% test item purity). An untreated control was also tested.

A 100 mg/L test item solution was prepared by adding a weighed amount of test item (ca 10 mg) to a 100 ml capacity volumetric flask and bringing to volume with ISO freshwater algal medium. This solution was sonicated for ca 10 min to aid dissolution. Aliquots of the 100 mg/L test item solution (0.2, 0.45, 1.0, 2.15 and 4.7 mL) were brought to volume with ISO freshwater algal medium in 500 ml volumetric flasks to prepare the 0.04, 0.09, 0.20, 0.43 and 0.94 mg/L treatment groups, respectively.

Flasks were prepared in triplicate for each treatment group, with six flasks prepared for the control. Each flask contained 100 mL of media and was inoculated with Pseudokirchneriella subcapitata. Algal cell concentrations were determined in each test flask at 24, 48, 72 and 96 h during the definitive test.

From visual observations and cell concentration determination it was determined that the 0.94 and 0.43 mg/L treatment groups had >50% inhibition of growth after 96 h exposure to WRS-2390TX. These were inoculated into fresh medium at the rate of 1:19 (exposure medium/fresh medium) and incubated for 9 days with the re-inoculated controls under similar conditions to the exposure phase Algal cell concentrations were determined in each test flask at 24, 48, 72 and 96 hours during the exposure phase of the definitive test and on Day 9 of the re-inoculation phase. Samples (2 mL) were removed from each flask and combined with 20 μL Lugol’s Iodine Solution. The samples were then shaken by hand, to ensure thorough mixing with the iodine, and stored at ca 4C until determination of cell concentration. Cell concentrations were determined within 7 days after sampling using a Compound Light Microscope and Improved Neubauer Counting Chambers.

An extra flask was prepared at the 0.94 and 0.09 mg/L treatment groups. These flasks were not inoculated with algal cells. Chemical analysis of the medium in these flasks, compared with the analysis of samples from inoculated flasks, indicate if adsorption/absorption to the algal cells occurred over the exposure period. The pH of the prepared test item stock solutions were determined at the beginning of the definitive test. In addition, the pH of every test flask was determined after the 96 h exposure phase.

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The 96 h EC50 for WRS-2390TX, based on Area Under the Curve (AUC), was determined to be 0.07 mg/L (measured)

Effect concentrations 2

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The No Observed Effect Concentration at 96h based on Area Under the Curve (AUC) was <0.02 mg/L,

Details on results:

- Exponential growth in the control (for algal test): Yes

Results with reference substance (positive control):

nda

Reported statistics and error estimates:

The 72 h EC50 value for WRS-2390TX, based on the area under the curve (AUC), was determined to be 0.06 mg/L.
The 72 h EC50 value for WRS-2390TX, based on growth rate, was determined to be 0.13 mg/L.
The No Observed Effect Concentration (NOEC) at 72h was 0.02 mg/L, based on AUC and <0.02 mg/L based on growth rate.
The 96 h EC50 value for WRS-2390TX, based on the area under the curve (AUC), was determined to be 0.07 mg/L.
The 96 h EC50 value for WRS-2390TX, based on growth rate, was determined to be 0.16 mg/L.The No Observed Effect Concentration (NOEC) at 96h was <0.02 mg/L, based on AUC and 0.11 mg/L based on growth rate.
The EC50 value for AUC at 72 h and Growth Rate at 24, 48, and 72 h are lower than the 96 h values. This is attributed to the growth pattern observed during the test.

Any other information on results incl. tables

The cell density data was used to calculate Area Under the Curve (AUC) biomass estimates and growth rate estimates. These were used in the statistical analysis to give end points for the exposure phase.

No growth was seen at the highest concentration (0.61 mg/L). The 0.27 mg/L cultures were growing for the first 48 h followed by a reduction in biomass. Growth in the control cultures was strong over the 96 h period.

The key concentration appeared to be the 0.11 mg/L test group. For this concentration the highest rate of inhibition for both measures occurred within the first 24 to 48 h. Data at 96 h, for this concentration, was the least inhibited time point. This suggests that these cultures recovered from initial exposure to the test item. It was likely this was linked to the apparent adsorption/absorption indicated in the analysis.

Algal cells may have reduced the concentration of available test item, allowing new cells to grow and the culture to recover. Results show measured concentrations at the initiation of the test at 0.14 mg/L whereas this was reduced to 0.09 mg/L after 96 h (a ca 36 % reduction in concentration) which was lower than the NOEC for growth rate determined in this test.

The pH recorded during the definitive test pH increased by >1 unit in all flasks where algal growth was observed. This is attributed to algal growth and did not have an impact on the outcome of the study.

The temperature recorded continuously during the definitive test was in the range 22.7-23.8 °C.

Based on statistical analysis of the cell numbers observed after the 9 day re-inoculation phase no significant differences were found in growth rate. This indicated that the effect of the test item to algal cells was phytostatic and not phytotoxic. However, the cells in the test item treated flasks were noted to have formed clumps visible to the naked eye whereas growth in the control flasks was normal.

Validity of the Test.

The cell density of control cultures increased by a factor of ca. 140 which greatly exceeded the ≥16 required in the protocol. The pH of the control cultures in 5 of the 6 replicates increased by up to 2.5 pH units over the 96 h period. However, based on the growth data, this was not thought to have affected the outcome of the study.

The coefficient of variation in daily growth in the control cultures was < 35% in line with protocol requirements. The coefficient of variation of average growth exceeded 15 % in all control replicates, which is a deviation from the protocol. However, Figure 2 (attachments) indicated that logarithmic growth was achieved in control cultures after a 24 h lag period, in line with the OPPTS Guideline. Hence this study is reported and the results regarded as acceptable.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See "Any other information on results incl. tables section", above.

Conclusions:

For the green alga Pseudokirchneriella subcapitata, the 96 hour EC50 value of WRS-2390TX based on growth rate was calculated to be 0.16 mg/L
(measured). The No Observed Effect Concentration at 96 h based on growth rate was 0.11 mg/L. Statistical analysis of daily cell counts was conducted. The 96 h EC50 for WRS-2390TX, based on Area Under the Curve (AUC), was determined to be 0.07 mg/L (measured).

The No Observed Effect Concentration at 96h based on AUC was <0.02 mg/L, the lowest geometric mean measured concentration tested.
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability & repeatability).

Executive summary:

In an Algae, Growth Inhibition Test (Inveresk report number: 23966) WRS-2390TX was found to have an NOEC of 0.11 mg/L.

For the green alga Pseudokirchneriella subcapitata, the 96 hour EC50 value of WRS-2390TX based on growth rate was calculated to be 0.16 mg/L. The 96 h EC50 for WRS-2390TX, based on Area Under the Curve (AUC), was determined to be 0.07 mg/L (measured).

The No Observed Effect Concentration at 96h based on AUC was <0.02 mg/L, the lowest geometric mean measured concentration tested whereas the NOEC was 0.11 mg/L (measured) based on the growth rate

The effect of the test item to algal cells was phytostatic and not phytotoxic, as no significant differences were found in growth rate after 9 days in fresh untreated medium. However the cells in the two test item treatments were both noted to have formed clumps visible to the naked eye, compared to the controls where growth was normal. Statistical analysis of daily cell counts was conducted.

.

The method was determined according to EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) Guideline and in compliance with the OECD principles of Good Laboratory Practice.