N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

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:

; analytical quantification of the test concentrations was not possible due to the absence of a suitable analytical method. Therefore only the stock solution used to prepare the test concentrations were analyzed.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Lot/Batch number: FP 020010155
- Triamine content: 85.3%; Total amine content (commercial grade): 100%

Analytical monitoring:

yes

Details on sampling:

Cell concentrations were determined photometrically with a UV/VIS Spectrophotometer (Standard Operation Procedure K 3 (9.11)). Measurements were carried out at 436 nm in a cuvette with a light path of 4 cm. To establish the relation between extinction and cell number/algal biomass, a calibration curve was made which is checked yearly. The procedure of constructing the calibration curve as well as the control procedure are described in Standard Operation Procedure E 3 (9.10).
From the relation between extinction (E) and counted cell number (N) the following calibration curve was determined using linear regression: N = 3.2034 * 10^6 * Extinction - 3.0 * 10^5 (r" = 0.9864).

The available analytical method was not suitable to quantify concentrations below 10 mg/L. Therefore only the stock solution used to prepare the test concentrations was analyzed. The measured concentration of the undiluted stock solution (1080 mg/L) differs only slightly from the nominal concentration of the stock solution (1066 mg/L). These results show that the stock solution was stable even after storage for almost 7 months in the refrigerator. All concentrations mentioned in this report are therefore based on nominal concentrations.

Vehicle:

no

Details on test solutions:

STOCK AND TEST SOLUTION AND THEIR PREPARATION:
- A stock solution of approximately 1 g/L of test substance was prepared as follows: To an accurately measured amount of 0.1066 g. of test substance some OECD medium of 51.3°C was added. The test substance dissolved well under stirring. After cooling, OECD medium was added to a final volume of 100 mL. A homogeneous, clear stock solution was obtained. The pH was 8.4. It was kept stirring at room temperature before being used for the preparation of the test solutions at the start of the test.

- The test solutions were prepared by addition of the required amounts of a 1:100 diluted stock solution to the test vessels to obtain the following final nominal test concentrations: 0.01, 0.02, 0.04, 0.08 and 0.16 mg/L.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISMS (Fresh water algae):
- Strain: Pseudokirchneriella subscapitata (was formally called Selenastrum capricornutum)
- Source: Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, The Windermere Laboratory, Cumbria, Ambleside, United Kingdom.
- Laboratory culture: Yes
- Method of cultivation: According to OECD 201
- Pretreatment: None
- Initial cell concentration: 1*E04 cells/mL
- Other: 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 below. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1*E04 cells/mL in the test medium.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.4-23.6 °C

pH:

8.2-8.7

Salinity:

Standard OECD 201 medium

Nominal and measured concentrations:

Nominal test concentrations: 0.01, 0.02, 0.04, 0.08 and 0.16 mg/L.

Details on test conditions:

TEST SYSTEM:
- Test vessel: 100 mL erlenmeyers containing 40 mL medium
- Renewal of test solution: No
- Number of replicates: 6 control vessels and 3 vessels per test substance concentration
- Test type: growth inhibition, endpoint biomass


TEST MEDIUM / WATER PARAMETERS:
- Test medium: The test medium (OECD medium) was prepared from concentrated solutions of the mineral salts prepared in deionized water. 3 L was prepared for the test and sterilized by filter sterilization (0.2 um).
- Macro nutrients (mg/L): NH4Cl; 15 KH2PO4; 1.6 CaCl2(H2O)2; 18 MgSO4(H2O)7; 15 MgCl2(H2O)6; 12 Fe-EDTA FeCl2(H2O)6; 0.08 Na2H2EDTA(H2O)2; 0.1 - Trace elements: H3BO3; 0.185 ZnCl2; 0.003 MnCl2(H2O)4; 0.415 CoCl2(H2O)6; 0.0015 CuCl2(H2O)2; 1*10-5 Na2MoO4(H2O)2; 0.007 NaHCO3 NaHCO3 150
- For the test an adequate amount of test medium was prepared in a 3-L vessel. This medium was sterilized by filter sterilization (0.2 μm). Adequate amounts of stock solution were added to the test vessels (100 mL erlenmeyers). The test vessels were filled with medium up to a total volume of 40 mL using a sterilized dispenser. For the concentrations 0.01 - 0.04 and 0.16 mg/L; 1L of test solution was prepared from which three times 40 mL was divided in the 100 mL test vessels. The remaining 880 mL was divided up in two portions of 440 mL from which one was stored for chemical analyses and one was transferred into a 1 L erlenmeyer. The inoculum was added to all 100 mL test vessels from an exponentially growing culture with a pipette. In addition six control replicates were included. The extinction in each 100 mL erlenmeyer was measured after 0, 24, 48 and 72 h. Algal medium was used as a blank in the spectrophotometer. The 1 L erlenmeyers with 440 mL 0.01, 0.04 and 0.16 mg/L were incubated together with the 100 mL test vessels in the orbital incubator.
- 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 below. From this algal culture a dilution was prepared to obtain an initial cell density of approximately 1*E04 cells/mL in the test medium.


TEST SOLUTION, CONTROLS AND TEST CONDITIONS:
- Dilution water: The deionized water used in the study contained not more than 0.01 mg/L of copper and had a TOC content of not more than 2.0 mg/L and a conductivity of less than 5 uS/cm. This water was produced from tap water in a water purification system.
- The test solutions were prepared by addition of the required amounts of a 1:100 diluted stock solution to the test vessels to obtain the following final nominal test concentrations: 0.01, 0.02, 0.04, 0.08 and 0.16 mg/L (nominal).
- Controls containing only test medium were included in the test.
- The vessels were incubated in an illuminated and temperature controlled (23°C ± 2 °C) orbital incubator.
- Aeration: No
- The test vessels were rotated continuously at a speed sufficient to prevent sedimentation of the algae. The extinction in each 100 mL erlenmeyer was measured after 0, 24, 48 and 72 h. Algal medium was used as a blank in the spectrophotometer. At the end of the test five random samples were microscopically checked for purity of the algal culture.
- Intensity of irradiation: 88-89 umol/m-2s-1
- Photoperiod: Continuous
- The pH of all samples and controls were measured at the beginning (t=0 h) and at the end (t=72 h) of the test. The temperature in the culturing apparatus was continuously measured and read out at the end of the test. The light intensity was measured at the beginning and at the end of the test. At the end of the test five random samples were microscopically checked for purity of the algal culture.
- Cell concentrations were determined photometrically with a UV/VlS Spectrophotometer. Measurements were carried out at 436 nm in a cuvette with a light path of 4 cm. To establish the relation between extinction and cell density, a calibration curve was made which was checked yearly. From the relation between extinction (E) and counted cell number (N) the following calibration curve was determined using linear regression: N= 3.2034*106 E- 3.0*105 (r2 = 0.9864).


STABILITY OF THE TEST CHEMICAL SOLUTIONS:
- Stock solution were stable after 7 months in refrigerator

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

other: EbC50

Effect conc.:

0.01 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Based on total amine content

Basis for effect:

biomass

Remarks on result:

other: (0.012-0.020 95% C.I.)

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

0.015 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Based on total amine content

Basis for effect:

growth rate

Remarks on result:

other: (0.012-0.020 95% C.I.)

Key result

Duration:

72 h

Dose descriptor:

other: ErC10

Effect conc.:

0.009 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Based on total amine content

Basis for effect:

growth rate

Remarks on result:

other: (0.0045-0.0121 95% C.I.)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.01 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Based on total amine content

Basis for effect:

biomass

Remarks on result:

other:

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.01 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Results with reference substance (positive control):

The sensitivity of the algae is checked by performing a growth inhibition test with a reference compound (potassium dichromate) twice a year, when the cultures are grafted from the sloped agar tubes. The sensitivity was tested for compliance with the Guidelines, which means that the EC50 values are between 0.25 and 2.0 mg/L.

Reported statistics and error estimates:

The mean values of the extinction’s for each test substance concentration were used to calculate the growth and specific growth rate.
Where possible, the EC20,50,80 values were computed from the best fitted line (least-squares method) through the points given by the probit of the percentage of inhibition and the logarithm of the concentration of the test substance. The EC50 value calculated for the area under the growth curve is termed EbC50 (0 72 h), whereas the EC50 value calculated for the specific growth rate is termed ErC50. The Lowest Observed Effect Concentration (LOEC) was determined by comparison of the growth at each concentration and the control using threshold values from the William’s test. The No Observed Effect Concentration (NOEC) was derived from the results as the first concentration below the LOEC value, where growth shows no significant inhibition relative to the control values. Confidence limits were computed on the basis of Fieller's theorem. All computations were performed using the TOXCALC version 5.0 program

RESULTS: EXPOSED- Nominal/measured concentrations: 0.01, 0.02, 0.04, 0.08, 0.16 mg/L (nominal).
- Effect data/Element values: 
Table 1: Result details:
Test-Substance
Concentration
(nominal)                               Inhibition%  
[mg/L]                                  compared to
                               0 h                   24 h                  48 h                    72 h              control 
0.00	0.008	0.038	0.157	0.654	0.0
0.01	0.008	0.018	0.075	0.346	12.7
0.02*	0.008	0.004	0.008	0.022	73.9
0.04	0.007	0.002	0.001	0.003	114.3
0.08	0.007	0.001	0.002	0.001	125.0
0.16	0.007	0.001	0.001	0.001	132.5
*One value was excluded from the calculations because it was an outlier (Grubbs outlier test). 
Validity criteria:    

Validity criteria were considered as fulfilled.

- The pH measurements a maximum increase of 0.5 pH units.

- The temperature varied from 23.4 to 23.6°C during the test, and the light intensity varied between approximately 88 and 89 umol/m-2s-1, which are both in accordance with the required conditions described in the study plan.

- The five random samples taken at the end of the test were all pure and not contaminated with bacteria.

- The increase factor of cell growth was 87 over 72 h for the controls.

- A dose-response related increase in growth inhibition observed.

The pH measurements a maximum increase of 0.5 pH units. The temperature varied from 23.4 to 23.6°C during the test, and the light intensity varied between approximately 88 and 89 μmol/m-2s-1, which are both in accordance with the required conditions described in the study plan. The five random samples were all pure and not contaminated with bacteria.

ALGAL INHIBITION TEST                                                                                                   

           PROGRAM: ALGAL2A                                                                                             

           Project:T01014AL Y12D                                                                                            

Concentration              Time (hours)              Area    Inhibition        Specific          Inhibition

(mg/L) 0         24       48       72                                   (%)           Growth rate        (%)

0.000  0.008  0.039  0.185  0.717             13.500                       0.063  

0.000  0.007  0.039  0.173  0.657             12.552                       0.063  

0.000  0.008  0.039  0.167  0.676             12.576                       0.062  

0.000  0.007  0.039  0.159  0.690             12.612                       0.063  

0.000  0.008  0.036  0.131  0.604             10.776                       0.059  

0.000  0.008  0.034  0.124  0.579             10.260                       0.059  

Mean  0.008  0.038  0.157  0.654             12.046                       0.062             0.0

                                                                                                                     

0.010  0.008  0.021  0.083  0.409              6.924                        0.055  

0.010  0.008  0.016  0.066  0.337              5.532                         0.053  

0.010  0.008  0.016  0.076  0.292              5.232                         0.052  

Mean  0.008  0.018  0.075  0.346              5.896             51.1     0.054             12.7

                                                                                                                     

0.020  0.007*0.036* 0.140*0.594*            Outlier                       Outlier

0.020  0.008  0.003  0.009  0.027             0.132                         0.020  

0.020  0.008  0.005  0.007  0.017             0.012                         0.011  

Mean  0.008  0.004  0.008  0.022             0.072             99.4     0.016             73.9

                                                                                                                     

0.040  0.007  0.001  0.001  0.002              -0.348                        -0.013 

0.040  0.007  0.001  0.001  0.003              -0.336                        -0.009 

0.040  0.007  0.003  0.002  0.005              -0.240                        -0.005 

Mean  0.007  0.002  0.001  0.003             -0.308            102.6   -0.009            114.3

                                                                                                                     

0.080  0.007  0.001  0.002  0.002              -0.324                        -0.011 

0.080  0.007  0.001  0.001  0.001              -0.360                        -0.019 

0.080  0.007  0.001  0.002  0.001              -0.336                        -0.017 

Mean  0.007  0.001  0.002  0.001              -0.340            102.8   -0.015            125.0

                                                                                                                     

0.160  0.007  0.001  0.001  0.000              -0.372                        -0.031 

0.160  0.007  0.002  0.000  0.002              -0.348                        -0.019 

0.160  0.007  0.001  0.001  0.001              -0.360                        -0.019 

Mean  0.007  0.001  0.001  0.001             -0.360            103.0   -0.021            133.8

The coefficient of average specific growth rates (72 hours test)
Time (hours)			mean	SD ±	%CV	Mean
0-24	24-48	48-72	72 hours test			%CV
0.066	0.065	0.056	0.0624	0.00522	8.36	9.35
0.072	0.062	0.056	0.0631	0.00803	12.73
0.066	0.061	0.058	0.0616	0.00397	6.45
0.072	0.059	0.061	0.0638	0.00689	10.80
0.063	0.054	0.064	0.0601	0.00543	9.03
0.060	0.054	0.064	0.0595	0.00520	8.74
		mean	0.0617
		stdev	0.0017
		CV%	2.7455

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h EbC50 and ErC50, based on nominal concentrations were 0.010 and 0.015 mg/L, respectively. The LOEC was determined to be at the lowest tested concentration of 0.010 mg/L. The ErC10 of 0.0095 mg/L will be used as long term endpoint.

Executive summary:

A study was conducted to determine the acute toxicity of the test substance to the freshwater algae Pseudokirchneriella subcapitata according to OECD Guideline 201, EU Method C.3 and ISO Method 8692, in compliance with GLP. In this study, triplicate Erlenmeyers containing the freshwater algae were exposed to 0.01, 0.02, 0.04, 0.08 and 0.16 mg/L nominal concentrations of the test substance for 72 h under static conditions. Biomass and growth rate were determined after 0, 24, 48 and 72 h. Although quantification of the applied concentrations was not possible due to the absence of a suitable analytical method it was possible to analyze the two stock solutions used to prepare the test concentrations. These analyses showed that the stock solutions were stable even after storage for almost 7 months in the refrigerator. Considering the high water solubility and stability of the test substance, it was considered reasonable to assume that the actual concentrations would be identical to the nominal concentrations during the entire test. All the validity criteria were considered to be fulfilled. Under the study conditions, the 72 h EbC50 and ErC50, based on nominal concentrations were 0.010 and 0.015 mg/L, respectively. The LOEC was determined to be at the lowest tested concentration of 0.010 mg/L. The ErC10 of 0.0095 mg/L will be used as long term endpoint (Geurts, 2002).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

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:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

For the purpose of the definitive study, the test material was dissolved directly in culture medium.
An amount of test material (100 mg) was dissolved in culture medium with the aid of ultrasonication (approximately 5 minutes) and the volume adjusted to 500 mL to give a 200 mg/L
stock solution. A series of dilutions were made from this stock solution to give further stock solutions of 20, 2.0, 0.20, 0.10, 0.050, 0.025 and 0.0125 mg/L. An aliquot (500 mL) of the 0.0125, 0.025, 0.050, 0.10 and 0.20 mg/L stock solutions was separately mixed with algal suspension (500 mL) to give the required test concentrations of 0.00625, 0.0125, 0.025, 0.050 and 0.10 mg/L

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test material in the test solutions were verified by chemical analysis at 0 and 72 hours.

Pre-study recovery analysis conducted indicated that the test material adsorbed to algal cells and hence it was considered that chemical analysis of the O-Hour test samples would show relatively low measured test concentrations. Therefore, in order to confirm the correct preparation of the initial stock solution and subsequent dosing of the test preparations, samples were prepared in culture medium alone (no algal cells) at 0 hours and taken for chemical analysis.

Analysis of the test solutions containing no algal cells showed measured test concentrations to be in the range of 87% to 127% of nominal thereby indicating that the correct dosing procedures were followed.

Chemical analysis of the test solutions containing algal cells at 0 hours showed measured test concentrations to be in the range of 58% to 81 % of nominal. Analysis of the test solutions at 72 hours showed a marked decline in measured test concentrations to below the limit of quantitation (LOQ) which was assessed down to 0.0035 mg/L and 4% of nominal. These results were inline with the preliminary recovery analysis conducted, which indicated that the test material adsorbed to algal cells.
The pre-study stability analysis conducted indicated that the test material was stable in culture medium over the study period. Adsorption was not a factor in the pre-study stability analysis since no algal cells were present.
In order to give a worst case analysis of the data, it was considered justifiable to base the results on the time-weighted mean measured test concentrations. Following the recommendations of the competent authorities in the EU, in cases where the measured test concentrations were less than the LOQ, a value of half the LOQ was used for calculating the time-weighted mean measured test concentrations.

The use of the time-weighted mean measured test concentrations in the calculation of the EC50 and NOEC values had a significant effect on the results of the study. Care should be taken in the interpretation of the results based on the time-weighted mean measured test concentrations as the test material has been shown to adsorb to algal cells.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test was carried out using Scenedesmus subspicatus strain CCAP 276/20. Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, The Ferry House, Far Sawrey, Ambleside, Cumbria.
Cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.2). The culture was maintained in the laboratory at a temperature of 21 ± 1°C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1°C.

pH:

7.3 - 10.5

Salinity:

Standard OECD 201 test medium

Nominal and measured concentrations:

See table below

Details on test conditions:

- Scenedesmus subspicatus was exposed to a geometric series of five test concentrations and a negative control. The study was conducted in glass conical flasks plugged with polyurethane foam bungs to reduce evaporation.
- Three replicate flasks were prepared for each test concentration and control. The nominal cell density in flasks at the initiation of the study was l0 cells per ml. Flasks were incubated under continuous light and constant shaking for 72 h.
- Samples were taken at 0, 24, 48 and 72 h and cell densities determined using a particle counter.
- pH was recorded at initiation and termination of the study.
- Temperature was recorded daily.
- Duplicate samples for verification of test concentrations were taken at 0 and 72 h and stored frozen.
- Inhibition of growth was calculated from comparison of area under the growth curves of the test concentration versus the control.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.012 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Remarks:

Based on total amine content

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Remarks:

Based on total amine content

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.007 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Remarks:

Based on total amine content

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.012 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Remarks:

Based on total amine content

Basis for effect:

biomass

Details on results:

- At test initiation the control and test cultures were clear colourless solutions.
- At the 72 h observation, the control, 0.00625, 0.0125, 0.025 and 0.050 mg/L test cultures were bright green dispersions and the 0.1 mg/L test culture was a very pale green dispersion.
- Analysis of the test concentrations showed a decline in test concentration in solutions containing algal cells. At 0 h concentrations ranged from 58 to 81 % of nominal and at 72 h test concentrations were below the limit of quantification to 4 % of nominal. Adsorption to algal cells was considered to be the cause of the decline in test substance concentration. This was confirmed by measuring the test concentrations in test vessels not containing algae (measured test concentrations were within 87 to 127 % of the nominal concentrations).

- The pH values of the control cultures were observed to pH 7.3 at 0 h to pH 10.4 -10.5 at 72 h. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution, which results in an increase in the pH of the culture. The increase in pH after 72 h was in excess of that recommended in the EEC Guidelines (1.5 pH units after 72 h). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the test guideline.

- Analysis of test solutions showed measured test concentrations to be in the range of 58 % to 81 % of nominal at 0 h and to below the LOQ at 72 h. To confirm the loss was due to adsorption to algal cells, samples were prepared in culture medium alone. Analysis of these solutions showed test concentrations in the range of 87 to 127 % of nominal. Pre-stability analysis showed that the test material was stable in the culture medium over the study period.

These factors are not considered to have affected the validity of the study.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 72 h EbC50 and ErC50 of the test substance, based on measured concentrations, were 0.012 and 0.02 mg/L, respectively. The NOEC and ErC10 were determined at 0.0069 and 0.012 mg/L, respectively.

Executive summary:

A study was conducted to determine the acute toxicity of the test substance to the freshwater algae Scenedesmus subspicatus according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Based on the results of two range finding studies, the algae were exposed to 0.00625, 0.0 125, 0.025, 0.050, 0.10 mg/L nominal concentrations of the test substance under static conditions for 72 h. Biomass and growth rate were determined after 0, 24, 48 and 72 h. Analysis of the test concentrations showed a decline due to adsorption to the algal cells. At 0 h, concentrations ranged from 58 to 81% of nominal values; at 72 h, the concentrations ranged from below the limit of quantification to 4% of nominal. Under the study conditions, the 72 h EbC50 and ErC50 of the test substance, based on measured concentrations, were 0.012 and 0.02 mg/L, respectively. The NOEC and ErC10 were determined at 0.0069 and 0.012 mg/L, respectively (Mead, 2001, 2007).

Key value for chemical safety assessment

EC50 for freshwater algae:

0.015 mg/L

EC10 or NOEC for freshwater algae:

0.009 mg/L

Additional information

A study was conducted to determine the acute toxicity of the test substance to the freshwater algae Pseudokirchneriella subcapitata according to OECD Guideline 201, EU Method C.3 and ISO Method 8692, in compliance with GLP. In this study, triplicate Erlenmeyers containing the freshwater algae were exposed to 0.01, 0.02, 0.04, 0.08 and 0.16 mg/L nominal concentrations of the test substance for 72 h under static conditions. Biomass and growth rate were determined after 0, 24, 48 and 72 h. Although quantification of the applied concentrations was not possible due to the absence of a suitable analytical method it was possible to analyze the two stock solutions used to prepare the test concentrations. These analyses showed that the stock solutions were stable even after storage for almost 7 months in the refrigerator. Considering the high water solubility and stability of the test substance, it was considered reasonable to assume that the actual concentrations would be identical to the nominal concentrations during the entire test. All the validity criteria were considered to be fulfilled. Under the study conditions, the 72 h EbC50 and ErC50, based on nominal concentrations were 0.010 and 0.015 mg a.i./L, respectively. The LOEC was determined to be at the lowest tested concentration of 0.010 mg a.i./L. The ErC10 of 0.0095 mg a.i./L will be used as long term endpoint(Geurts, 2002).

A study was conducted to determine the acute toxicity of the test substance to the freshwater algae Scenedesmus subspicatus according to OECD Guideline 201 and EU Method C.3, in compliance with GLP. Based on the results of two range finding studies, the algae were exposed to 0.00625, 0.0 125, 0.025, 0.050, 0.10 mg/L nominal concentrations of the test substance under static conditions for 72 h. Biomass and growth rate were determined after 0, 24, 48 and 72 h. Analysis of the test concentrations showed a decline due to adsorption to the algal cells. At 0 h, concentrations ranged from 58 to 81% of nominal values; at 72 h, the concentrations ranged from below the limit of quantification to 4% of nominal. Under the study conditions, the 72 h EbC50 and ErC50 of the test substance, based on measured concentrations, were 0.012 and 0.02 mg a.i./L, respectively. The NOEC and ErC10 were determined at 0.0069 and 0.012 mg a.i./L, respectively (Mead, 2001, 2007).

A study was conducted to determine effects of the test substance on the freshwater algae Pseudokirchneriella subcapitata according to EPA OTS Guideline 797.1050, in compliance with GLP. No analytical dose verification was conducted. The effects on algal growth were algistatic rather than algicidal. Under the study conditions, the nominal 96 h EC50 and NOEC values for growth rate were determined to be 0.054 and 0.012 mg/L, respectively (Thompson, 1992).