Quaternary ammonium compounds, di-C8-10-alkyldimethyl, chlorides

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)

Version / remarks:

Also in accordance with US EPA OPPTS Number 850.5400

Deviations:

no

Principles of method if other than guideline:

In addition to the standard guideline, the test duration was extended to 96 hours and after 96 hours a recovery phase was observed for 6 days.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test substance Bardac 2280 containing 81% of didecyldimethyl ammonium chloride (DDAC) as the active ingrediant was provided by Lonza Inc. In addition, radio-labelled 14C-DDAC was received from Wizard Laboratories Inc with a reported purity of 99.88%.

Analytical monitoring:

yes

Details on sampling:

The freshwater alga was exposed to a geometric series of six test concentrations and a negative control under static conditions for 96 hours. Three replicate test chambers were maintained in each treatment and control group. Two replicate test chambers were also maintained with the absence of algae to verify test concentrations. Samples were collected from each replicate at test initiation and approximately 24 hour intervals during the test to determine cell densities for the calculation of growth inhibition.

Vehicle:

no

Details on test solutions:

A stock solution was prepared using 1.0 mCi of radio-labelled 14C-DDAC with 100 ml of NANOpure water. When analysed (98% recovery) the mean concentration was 326,418 dpm. This was further diluted to create a secondary stock solution used to prepare the test solutions and corresponded to 3.5 ug of the radio-labelled DDAC for each litre of test solution. In addition, non-labelled DDAC was prepared to a concentration of 0.20 ug DDAC/ml. The test medium was then prepared with equal volumes of the two stock solutions to prepare the 4.7, 9.4, 19, 38, 75 and 150 ug/L nominal test solutions. Test solutions were mixed by inversion. The potential for DDAC to bind to glassware was minmised by pre-treatment of the glassware with DDAC concentrations that corresponded to each treatment group, e.g. the 150 ug/l vessels were pretreated with 150ug/l DDAC solutions for 10 minutes. Several non-GLP trials were performed prior to the test to undertand binding potential to glassware. It was found that DDAC remained available to algae when bound to the glassware, hence the corresponding test concentration was used for pre-treatment.

Test organisms (species):

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

Details on test organisms:

The freshwater alga Selenastrum capricornutum was the test organism with original cultures obtained from the University of Toronto and further cultured in the laboratory's own facility. Algal cells were obtained from a culture that had been actively growing for at least two weeks prior to the test. The culture medium included a stock nutrient solution prepared by the laboratory with purified well water. The pH was adjusted to 7.5 +/- 0.1 using 10% HCl, where appropriate and sterlised by filtration before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

An exposure duration of 96 hours was used.

Post exposure observation period:

Following the 96 hour exposure duration, a recovery phase was observed for six further days by dilution of the test substance to a concentration that would not cause inhibition. Cell densities were counted at the initiation of the recovery phase, day 3 and day 6.

Hardness:

No information reported

Test temperature:

Temperature ranged from 23.2 to 24.4 degrees C

pH:

pH ranged from 7.2 to 7.3

Dissolved oxygen:

No information reported

Salinity:

No information reported

Conductivity:

No information reported

Nominal and measured concentrations:

Nominal concentrations were 0, 4.7, 9.4, 19, 38, 75 and 150 ug/L The mean measured DDAC concentrations were 0, 3.3, 6.86, 14.3, 29.9, 62.6 and 129.5 ug/L. The mean percentage of the nominal concentrations ranged from 70 to 86%.

Details on test conditions:

Test chambers were sterilised 250 ml Erlenmeyer flasks pretreated with DDAC (for each respective treatment concentration) and plugged with foam stoppers. Each contained 100 ml of test solution or control medium. Each vessel was labelled and randomly positioned daily on a mechanical shaker in an environmental chamber designed to maintain the test temperature. The flasks were shaken continuously at 100 rpm. The algae were under 24 hours fluorescent lighting. Environmental conditions were monitored twice daily.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

27 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

11 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

26 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

13 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

27 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Exponential growth was achieved and passed the OECD Guideline 201 QC criterion of a 16 fold increase in growth over 72 hours. After 96 hours of exposure to DDAC, inhibition of cell density in the 3.3, 6.9, 14, 30, 63 and 130 ug/L treatment groups was -0.93, 6.6, 7.8, 58, 98 and 99% respectively relative to the negative control. The 24-hour NOEC for cell density was 3.3ug DDAC/L. The 48, 72 and 96-hour NOEC was 14 ug DDAC/L. The 24-hour NOEC for biomass was 3.3 ug DDAC/L. The 48, 72 and 96-hour NOEC for biomass was 14 ug DDAC/L. After 96 hours of exposure there was no difference in cell shape, size or colour between any of the treatment groups and control replicates. In addtion, there was no evidence of aggregation or flocculation of cells, nor adherence to the test chambers.

In the study, 63 and 130 ug/L treatment groups were maximally inhibited at the end of the 96 hours exposure. By day 6 of the recovery phase, sufficient growth was observed in all relicates to indicate recovery from exposure to the test substance.

Results with reference substance (positive control):

No information reported

Reported statistics and error estimates:

Statistical analyses were conducted using the SAS system for Windows version 8.0. Cell densities and the area under the growth curve were statistically analysed by non-linear regression versus concentration to determine EC10 and EC50 values, including 95% confidence limits. Cell density and area under the growth curve were evaluated for normality and homogeneity of variance 9p=0.05) using the Shapiro-Wilk's and Levene's tests, respectively, and were compared to the negative control using analysis of variance (ANOVA) and Dunnett's test (p=0.05).

A summary of the results is presented in the tables below:

72-hour ECx values expressed as DDAC equivalents (µg/l)	Cell density	Growth inhibition
EC10 (95% confidence limits)	11 (8.2 to 16)	11 (8.0 to 16)
EC50 (95% confidence limits)	27 (22 to 32)	27 (22 to 32)
96-hour ECx values expressed as DDAC equivalents (µg/l)
EC10 (95% confidence limits)	14 (9.0 to 21)	13 (9.2 to 18)
EC50 (95% confidence limits)	26 (21 to 33)	26 (22 to 32)

NOEC expressed as DDAC equivalents (µg/l)	Cell density	Growth inhibition
24 hour	3.3	3.3
48 hour	14	14
72 hour	14	14
96 hour	14	14

Validity criteria fulfilled:

yes

Conclusions:

In a 96-hour static toxicity study of Selenastrum capricornutum exposed to the test substance (DDAC or didecyldimethyl ammonium chloride) resulted in a 72-hour and 96-hour EC50 of 27 and 26 ug/L as a measured concentration, respectively, for the inhibition of algal growth. The corresponding NOEC was 14 ug/L (both durations) for growth inhibition.

Executive summary:

A toxicity study of the test substance DDAC exposed to the freshwater algae Selenastrum capricornutum in a static test resulted in a 72-hour EC50 of 27 ug/L (0.027 mg/L) based on a growth inhibition endpoint. A NOEC of 14 ug/L (growth inhibition) was observed. The study was extended to observe recovery following exposure to the test substance and based on growth the algae were seen to recover. The reliable study corresponds to the OECD Test Guideline 201 and was undertaken by a commercial laboratory using its own algal culture. No adverse effects was observed in the control groups of organisms.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to EU- and/or OECD-guidelines.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Safepham Laboratories Ltd

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours.

Details on test solutions:

Identity and concentration of auxiliary solvent for dispersal: For the purpose of the definitive test, the test material was dissolved directly in culture medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 276120

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 +/- 1 °C

pH:

7.3 - 7.8

Nominal and measured concentrations:

Nominal concentrations: 0, 0.050, 0.10, 0.20, 0.40 and 0.80 mg/L
Measured concentrations: 0, 0.0191, 0.0502, 0.152, 0.353 and 0.798 mg/L

Details on test conditions:

The test was conducted in 250 mL glass conical flasks plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. The test material was dissolved directly in culture medium.
Scenedesmus subspicatus was exposed to an aqueous solution of the test material at concentrations of 0.050, 0.10, 0.20, 0.40 and 0.80 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 +/- 1 °C. At 0, 24, 48 and 72 hours, a sample was removed and the cell density determined.
Preliminary work suggested that the test material adsorbed to glassware and hence all glassware used in the definitive test was pre-conditioned with the test concentration to be used in the test, approximately 24 hours prior to the start of the test. The preliminary work also suggested that the test material may adsorb to algal cells and hence a further set of test concentrations was prepared at 0 hours in culture medium to confirm correct dosing.

Reference substance (positive control):

not required

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.022 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 0.020 – 0.026 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.035 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 0.029 – 0.042 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.015 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Details on results:

All test and control cultures were inspected microscopically at 72 hours and there were no abnormalities detected in the control or test cultures at 0.050, 0.10 and 0.20 mg/L, however few intact algal cells were observed to be present in the test cultures at 0.40 and 0.80 mg/L.
Chemical analysis of the test preparations at 0 hours showed test concentrations to be in the range of 81 – 105 % of nominal with the exception of the 0.050 mg/L test samples, which were 45 % of nominal in the sample that contained algal cells and 69 % of nominal in the sample prepared in culture medium alone.
Given that there was a decline in measured test concentrations over the test period, it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.
The low result in culture medium alone was considered to be due to further adsorption of the test material to the glassware used, despite being pre-conditioned. The result obtained with algal cells was considered to be the result of a combination of adsorption to glassware and algal cells.
Analysis of the test preparations at 72 hours showed a decline in measured test concentrations with values in the range of 29 – 97 % of nominal. Overall the decline in measured concentrations followed a concentrations dependent pattern with greater losses being observed at the lower test concentrations. This effect was considered to be due to there being greater numbers of algal cells in the lower concentrations and hence greater surface area for adsorption to occur.

Table 1: Inhibition of the growth and the growth rate

Nominal concentration (mg/L)	Area under curve at 72 h	% Inhibition	Growth rate (0-72h)	% Inhibition
Control	5.04E^6	-	0.047	-
0.050	4.84 E^6	4	0.049	[4]
0.10	8.16 E^6	84	0.015	68
0.20	2.13 E^6	100	0.000	100
0.40	-1.14 E^5	102	-0.019	140
0.80	-3.31 E^5	107	-0.034	172

[Increase in growth as compared to controls]

Validity criteria fulfilled:

yes

Conclusions:

In terms of the pure active ingredient (as 100 % AI) 72-hour EbC50 was determined 0.022 mg/L (0.020 – 0.026 mg/L), the 72-hour ErC50 was determined 0.035 mg/L (0.029 – 0.042 mg/L) and the No Observed Effect Concentration (NOEC) at 72 hours 0.0152 mg/L.

Executive summary:

A study was carried out according to EU Method C.3 and OECD Guideline 201 (Alga, Growth Inhibition Test). Following preliminary range-finding testing, Scenedesmus subspicatus was exposed to an aqueous solution of the test material at mean measured concentrations of 0, 0.0191, 0.0502, 0.152, 0.353 and 0.798 mg AI/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 +/- 1 °C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Panicle Counter.

Preliminary work suggested that the test material absorbed to glassware and hence all glassware used in the definitive test (test vessels and sample bottles) was pre-conditioned with the test concentration to be used in the test, approximately 24 hours prior to the test start. The preliminary work also suggested that the test material may adsorb to algal cells and hence a further set of test concentrations was prepared at 0 hours in culture medium alone to confirm correct dosing procedures.

Chemical analysis of the test preparations at 0 hours showed measured lest concentrations to be in the range of 81 % to 105 % of nominal with the exception of the 0.050 mg AI/L test samples which were 45 % of nominal in the sample that contained algal cells and 69 % of nominal in the sample prepared in culture medium alone.

The relatively low result obtained for the 0.050 mg AI/L test sample prepared in culture medium alone was considered to be due to further adsorption of the test material to the glassware used.

Despite pre-conditioning the glassware for approximately 24 hours prior to the start of the test it was considered that there might not have been sufficient test material present at this concentration to saturate all adsorption sites. In the 0.050 mg AI/L test sample that contained algal cells the measured test concentration was 45 % of nominal, this low measured concentration was considered to be the result of a combination of adsorption to glassware and algal cells.

Analysis of the test preparations at 72 hours showed a decline in measured test concentrations with values in the range of 29 % to 97 % of nominal.

Given that the stability analyses showed that the test material was stable the decline in measured test concentrations was considered to be due to adsorption to algal cells and was in line with the recovery analyses performed with the addition of algal cells. Overall the decline in measured concentrations followed a concentration dependent pattern with greater losses being observed at the lower test concentrations. This effect was considered to be due to there being greater numbers of algal cells in the lower concentrations and hence greater surface area for adsorption 10 occur. Adsorption was not a factor in the stability analyses conducted as no algal cells were present.

Given that there was a decline in measured test concentrations over the test period, it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. In terms of the pure active ingredient (as 100 % AI) the 72-hour EbC50 was determined 0.022 mg AI/L (0.020 – 0.026 mg AI/L), the 72-hour ErC50 was determined 0.035 mg AI/L (0.029 – 0.042 mg AI/L) and the No Observed Effect Concentration (NOEC) at 72 hours 0.0152 mg AI/L.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to EU- and/or OECD-guidelines.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples of the test solutions were collected at approximately 0 and 96 hours to measure concentrations of the test substance. Samples at test initiation were collected from the individual batches of test solution prepared for each treatment and control group prior to addition of the algae. At exposure termination, samples were collected and pooled from the three replicates from each treatment and control group, respectively. All samples were collected and transferred into glass vials containing an equal volume of an organic solvent and were analyzed immediately without storage.

Vehicle:

no

Details on test solutions:

A primary stock solution was prepared by dissolving test item in saltwater algal medium at a nominal concentration of 41.5 mg AI/L. The stock was inverted at least 20 times to mix and appeared slightly cloudy and colorless with foam on the surface. A secondary stock was prepared by diluting 2.0 mL of the 41.5 mg AI/L stock solution to achieve the highest nominal test concentration 83 μg mg AI/L.
The secondary stock was inverted at least 20 times and appeared clear and colorless. The secondary stock was proportionally diluted with saltwater algal medium to prepare the four additional test solutions at nominal concentrations of 16, 24, 37 and 55 μg AI/L. Test concentrations were corrected for active ingredient in the test substance. All test solutions appeared clear and colorless.
Because test item has a tendency to bind to glassware, the glassware and test flasks used for each treatment group were pretreated for approximately 24 hours with solutions of test item prepared in order to minimize the binding of Test item to the sides of the glassware and test flasks.
The test item concentrations of pretreatment solutions matched the Test item concentrations of each treatment group. For example, the glassware used for the 83 μg AI/L Test item treatment group was pretreated with 83 μg AI/L test item solutions for approximately 24 hours.

Test organisms (species):

Skeletonema costatum

Details on test organisms:

The marine diatom, Skeletonema costatum (CCMP 1332), was selected as the test species for this study. The species is representative of an important group of diatoms, and was selected for use in the test based upon a past history of use, and ease of culturing in the laboratory. Original algal cultures were obtained from Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP), and had been maintained in culture medium at Wildlife International, Ltd., Easton, Maryland. Algal cells used in this test were obtained from Wildlife International, Ltd. cultures that had been actively growing in culture medium for at least two weeks prior to test initiation. The culture was last transferred to fresh medium four days prior to test initiation. The negative control organisms were expected to exhibit exponential growth over the 96-hour exposure period. Exponential growth phase, defined as the period of growth where the algal cells are dividing at a constant rate, is indicated by the linear section of the growth curve.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Aliquots of the 62 μg AI/L test solution was diluted with algal medium and cultured for 9 days.

Test temperature:

20 +/- 2 °C

pH:

8.1 - 8.5

Salinity:

Approx. 30 parts per thousand

Nominal and measured concentrations:

Nominal concentrations: 0, 16, 24, 37, 55 and 83 µg/L
Measured concentrations:
- Day 0: 0, 14, 20, 26, 40 and 62 µg/L
- Day 4: 0, 4.2, 7.1, 15, 31 and 48 µg/L

Details on test conditions:

The test was conducted in 250 mL Erlenmeyer flasks plugged with foam stoppers to reduce evaporation. The test material was dissolved directly in saltwater algal medium.
Skeletonema costatum was exposed to the test material at nominal concentrations of 16, 24, 37, 55 and 83 µg/L (three replicate flasks per concentration) for 96 hours, under constant illumination (4310 +/- 650 lux) and shaking at a temperature of 20 +/- 2°C. Samples collected on Day 0 gave measured concentrations as 14, 20, 26, 40 and 62 µg/L respectively. Samples taken after 96 hours gave measured concentrations as 4.2, 7.1, 15, 31 and 48 µg/L respectively. At 0, 24, 48, 72 and 96 hours, a sample was removed and the cell density determined.
Preliminary work suggested that the test material adsorbed to glassware and hence all glassware used in the definitive test was pre-conditioned with the test concentration to be used in the test, approximately 24 hours prior to the start of the test.

Reference substance (positive control):

not required

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

0.025 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 0.024 - 0.026 mg AI/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.02 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: NA

Details on results:

After 96 hours of exposure, cell density percent inhibition in the 14, 20, 26, 40 and 62 µg AI/L groups was – 0.16, 1.3, 64, 56 and 97 % respectively. Treatment related effects were apparent in the three highest concentrations. Cell density was statistically significantly reduced in the 26, 40 and 62 µg AI/L treatment groups.
After 96 hours of exposure, there were no signs of adherence of cells to the test chambers or aggregation/ flocculation in any of the treatment levels or the control. There were no noticeable changes in cell morphology in any of the tested concentrations when compared to the control.
The 62 µg AI/L treatment groups were maximally inhibited at the end of the 96 hour exposure period. Aliquots of the 62 µg AI/L test solution were diluted with algal medium and cultured for up to 9 days. However, based on the growth observed over the recovery phase, the effect on algal growth was found to be more algistatic than algicidal.
The pH of the test solutions at test initiation ranged from 8.1 to 8.2 and at exposure termination ranged from 8.1 to 8.5. The pH tended to increase relative to increase in algal densities, which is typical for tests conducted with Skeletonema costatum.

Validity criteria fulfilled:

yes

Conclusions:

After 96 hours of exposure, cell density percent inhibition in the 14, 20, 26, 40 and 62 μg AI/L treatment groups was - 0.16, 1.3, 64, 56 and 97 %, respectively, relative to the negative control. Treatment related effects were apparent in the three highest test concentrations. Dunnett’s test indicated that cell density was significantly reduced (p < 0.05) in the 26, 40 and 62 μg AI/L treatment groups. The 96-hour EC50 for cell density was determined to be 25 µg/L (24 – 26 µg/L), the 96-hour NOAEC was 20 μg AI/L.

Executive summary:

A study was carried out according to EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II). The marine diatom, Skeletonema costatum was exposed to nominal concentrations of 0, 16, 24, 37, 55 and 83 µg AI/L, corresponding to measured concentrations of 0, 14, 20, 26, 40 and 62 µg AI/L (Day 0) and 0, 4.2, 7.1, 15, 31 and 48 µg AI/L (Day 4).

After 96 hours of exposure, cell density percent inhibition in the 14, 20, 26, 40 and 62 μg AI/L treatment groups was 0.16, 1.3, 64, 56 and 97 %, respectively, relative to the negative control. Treatment related effects were apparent in the three highest test concentrations. Dunnett’s test indicated that cell density was significantly reduced (p < 0.05) in the 26, 40 and 62 μg AI/L treatment groups. The 96-hour EC50 for cell density was determined to be 25 µg/L (24 – 26 µg/L), the 96-hour NOAEC was 20 μg AI/L.

Aliquots of the 62 µg AI/L test solution were diluted with algal medium and cultured for up to 9 days. However, based on the growth observed over the recovery phase, the effect on algal growth was found to be more algistatic than algicidal.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other:

Reason / purpose for cross-reference:

read-across source

Specific details on test material used for the study:

Test substance Bardac 2280 containing 81% of didecyldimethyl ammonium chloride (DDAC) as the active ingrediant was provided by Lonza Inc. In addition, radio-labelled 14C-DDAC was received from Wizard Laboratories Inc with a reported purity of 99.88%.

Analytical monitoring:

yes

Details on sampling:

The freshwater alga was exposed to a geometric series of six test concentrations and a negative control under static conditions for 96 hours. Three replicate test chambers were maintained in each treatment and control group. Two replicate test chambers were also maintained with the absence of algae to verify test concentrations. Samples were collected from each replicate at test initiation and approximately 24 hour intervals during the test to determine cell densities for the calculation of growth inhibition.

Vehicle:

no

Details on test solutions:

A stock solution was prepared using 1.0 mCi of radio-labelled 14C-DDAC with 100 ml of NANOpure water. When analysed (98% recovery) the mean concentration was 326,418 dpm. This was further diluted to create a secondary stock solution used to prepare the test solutions and corresponded to 3.5 ug of the radio-labelled DDAC for each litre of test solution. In addition, non-labelled DDAC was prepared to a concentration of 0.20 ug DDAC/ml. The test medium was then prepared with equal volumes of the two stock solutions to prepare the 4.7, 9.4, 19, 38, 75 and 150 ug/L nominal test solutions. Test solutions were mixed by inversion. The potential for DDAC to bind to glassware was minmised by pre-treatment of the glassware with DDAC concentrations that corresponded to each treatment group, e.g. the 150 ug/l vessels were pretreated with 150ug/l DDAC solutions for 10 minutes. Several non-GLP trials were performed prior to the test to undertand binding potential to glassware. It was found that DDAC remained available to algae when bound to the glassware, hence the corresponding test concentration was used for pre-treatment.

Test organisms (species):

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

Details on test organisms:

The freshwater alga Selenastrum capricornutum was the test organism with original cultures obtained from the University of Toronto and further cultured in the laboratory's own facility. Algal cells were obtained from a culture that had been actively growing for at least two weeks prior to the test. The culture medium included a stock nutrient solution prepared by the laboratory with purified well water. The pH was adjusted to 7.5 +/- 0.1 using 10% HCl, where appropriate and sterlised by filtration before use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

An exposure duration of 96 hours was used.

Post exposure observation period:

Following the 96 hour exposure duration, a recovery phase was observed for six further days by dilution of the test substance to a concentration that would not cause inhibition. Cell densities were counted at the initiation of the recovery phase, day 3 and day 6.

Hardness:

No information reported

Test temperature:

Temperature ranged from 23.2 to 24.4 degrees C

pH:

pH ranged from 7.2 to 7.3

Dissolved oxygen:

No information reported

Salinity:

No information reported

Conductivity:

No information reported

Nominal and measured concentrations:

Nominal concentrations were 0, 4.7, 9.4, 19, 38, 75 and 150 ug/L The mean measured DDAC concentrations were 0, 3.3, 6.86, 14.3, 29.9, 62.6 and 129.5 ug/L. The mean percentage of the nominal concentrations ranged from 70 to 86%.

Details on test conditions:

Test chambers were sterilised 250 ml Erlenmeyer flasks pretreated with DDAC (for each respective treatment concentration) and plugged with foam stoppers. Each contained 100 ml of test solution or control medium. Each vessel was labelled and randomly positioned daily on a mechanical shaker in an environmental chamber designed to maintain the test temperature. The flasks were shaken continuously at 100 rpm. The algae were under 24 hours fluorescent lighting. Environmental conditions were monitored twice daily.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

27 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

11 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

26 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

13 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

27 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Exponential growth was achieved and passed the OECD Guideline 201 QC criterion of a 16 fold increase in growth over 72 hours. After 96 hours of exposure to DDAC, inhibition of cell density in the 3.3, 6.9, 14, 30, 63 and 130 ug/L treatment groups was -0.93, 6.6, 7.8, 58, 98 and 99% respectively relative to the negative control. The 24-hour NOEC for cell density was 3.3ug DDAC/L. The 48, 72 and 96-hour NOEC was 14 ug DDAC/L. The 24-hour NOEC for biomass was 3.3 ug DDAC/L. The 48, 72 and 96-hour NOEC for biomass was 14 ug DDAC/L. After 96 hours of exposure there was no difference in cell shape, size or colour between any of the treatment groups and control replicates. In addtion, there was no evidence of aggregation or flocculation of cells, nor adherence to the test chambers.

In the study, 63 and 130 ug/L treatment groups were maximally inhibited at the end of the 96 hours exposure. By day 6 of the recovery phase, sufficient growth was observed in all relicates to indicate recovery from exposure to the test substance.

Results with reference substance (positive control):

No information reported

Reported statistics and error estimates:

Statistical analyses were conducted using the SAS system for Windows version 8.0. Cell densities and the area under the growth curve were statistically analysed by non-linear regression versus concentration to determine EC10 and EC50 values, including 95% confidence limits. Cell density and area under the growth curve were evaluated for normality and homogeneity of variance 9p=0.05) using the Shapiro-Wilk's and Levene's tests, respectively, and were compared to the negative control using analysis of variance (ANOVA) and Dunnett's test (p=0.05).

A summary of the results is presented in the tables below:

72-hour ECx values expressed as DDAC equivalents (µg/l)	Cell density	Growth inhibition
EC10 (95% confidence limits)	11 (8.2 to 16)	11 (8.0 to 16)
EC50 (95% confidence limits)	27 (22 to 32)	27 (22 to 32)
96-hour ECx values expressed as DDAC equivalents (µg/l)
EC10 (95% confidence limits)	14 (9.0 to 21)	13 (9.2 to 18)
EC50 (95% confidence limits)	26 (21 to 33)	26 (22 to 32)

NOEC expressed as DDAC equivalents (µg/l)	Cell density	Growth inhibition
24 hour	3.3	3.3
48 hour	14	14
72 hour	14	14
96 hour	14	14

Validity criteria fulfilled:

yes

Conclusions:

In a 96-hour static toxicity study of Selenastrum capricornutum exposed to the test substance (DDAC or didecyldimethyl ammonium chloride) resulted in a 72-hour and 96-hour EC50 of 27 and 26 ug/L as a measured concentration, respectively, for the inhibition of algal growth. The corresponding NOEC was 14 ug/L (both durations) for growth inhibition.

Executive summary:

A toxicity study of the test substance DDAC exposed to the freshwater algae Selenastrum capricornutum in a static test resulted in a 72-hour EC50 of 27 ug/L (0.027 mg/L) based on a growth inhibition endpoint. A NOEC of 14 ug/L (growth inhibition) was observed. The study was extended to observe recovery following exposure to the test substance and based on growth the algae were seen to recover. The reliable study corresponds to the OECD Test Guideline 201 and was undertaken by a commercial laboratory using its own algal culture. No adverse effects was observed in the control groups of organisms.

Description of key information

A reliable study (Klimisch 1) for the read-across substance DDAC using the freshwater alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) is available (Desjardins, 2002). This study determined a 72-hour EC50 of 0.027 mg/L (27 ug/L) and a NOEC of 0.014 mg/L (14 ug/L) based on growth inhibition. The study was extended and growth was observed to recover. The study corresponds to the OECD Test Guideline 201 and was undertaken by a commercial laboratory using its own algal culture.

Two reliable studies (Klimisch 1) are available on the read-across substance N,N-Didecyl-N,N-dimethylammonium carbonate (DDACarbonate) (Safepharm, 2004; Wildlife International, 2004). In the Safepharm study (2004), the diatom, Skeletonema costatum, after 96 hours of exposure, resulted in a cell density percent inhibition in the 14, 20, 26, 40 and 62 μg AI/L treatment groups was 0.16, 1.3, 64, 56 and 97 %, respectively, relative to the negative control. The 96-hour EC50 for cell density was determined to be 25 µg/L (24 – 26 µg/L), the 96-hour NOAEC was 20 μg AI/L. Aliquots of the 62 µg AI/L test solution were diluted with algal medium and cultured for up to 9 days. However, based on the growth observed over the recovery phase, the effect on algal growth was found to be more algistatic than algicidal.

In the Wildlife International study (2004), OECD 201 test, Scenedesmus subspicatus was exposed to an aqueous solution of the read across substance DDACarbonate at mean measured concentrations of 0, 0.0191, 0.0502, 0.152, 0.353 and 0.798 mg AI/L. Stability analyses identified a decline in measured test concentrations throughout the test was likely due to adsorption to algal cells and was in line with the recovery analyses performed with the addition of algal cells. Overall the decline in measured concentrations followed a concentration dependent pattern with greater losses being observed at the lower test concentrations. Given that there was a decline in measured test concentrations over the test period, it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. In terms of the pure active ingredient (as 100 % AI) the 72-hour EbC50 was determined 0.022 mg AI/L (0.020 – 0.026 mg AI/L), the 72-hour ErC50 was determined 0.035 mg AI/L (0.029 – 0.042 mg AI/L) and the No Observed Effect Concentration (NOEC) at 72 hours 0.0152 mg AI/L.

The algal study data for DDAC and DDACarbonate (2 studies) determined ErC50 (growth rate inhibition) of 0.027 mg/L, 0.025 mg/L, 0.035 mg/L, respectively and NOEC’s of 0.014 mg/L, 0.020mg/L and 0.0152 mg/L, respectively. It is recommended that the algal study for DDAC (Desjardins, 2002) is the key study endpoint considered in PNEC development based on its reliability and the use of the test substance DDAC in toxicity testing

.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.122 mg/L

EC10 or NOEC for freshwater algae:

0.01 mg/L