Quaternary ammonium compounds, C12-14-alkyl(hydroxyethyl)dimethyl, chlorides

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:

1994-12-15 to 1995-03-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented, scientifically accepted methods.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

A flow-through laboratory microcosm using a proportional dilutor system was developed to assess effects of toxicants on algal periphyton communities. Artificial substrates were naturally colonized with periphyton by submersion in the upper reach of the Little Miami River, north of Xenia, Ohio, a high quality, National and State Scenic River. Colonized substrates were transported to the lab for dosing.

GLP compliance:

no

Remarks:

However, the final study report was audited by the ESD Quality Assurance Unit for accuracy and adherence to Departmental Standard Operating Procedures, the Study Protocol, and Environmental Protection Agency Good Laboratory Practice Regulations.

Test material

Test material information

Constituent 1

Reference substance name:

[see concordance under 1.1. of the CSR]

IUPAC Name:

[see concordance under 1.1. of the CSR]

Details on test material:

- Name of test material (as cited in study report): Alkyl dimethyl ethanol ammonium chloride (ADEAC) (KDB-Base) (E4408.02) This material is also known as C12-14 dimethyl hydroxyethyl ammonium chloride.
- Molecular formula (if other than submission substance): N/A
- Molecular weight (if other than submission substance): N/A
- Smiles notation (if other than submission substance): N/A
- InChl (if other than submission substance): N/A
- Structural formula attached as image file (if other than submission substance): N/A
- Substance type: cationic surfactant
- Physical state: liquid

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: N/A
- Boiling point: N/A
- Vapour pressure: N/A
- Water solubility (under test conditions): N/A
- Henry's law constant: N/A
- log Pow: N/A
- pKa: N/A
- Stability in water: N/A
- Stability in light: N/A
- pH dependance on stability: N/A


OTHER PROPERTIES (if relevant for this endpoint)
- Results of test for ready biodegradability: N/A
- Other: N/A

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 12.5, 25, 50, 100, and 200 micrograms/L (nominal)
- Sampling method: Samples were obtained near the effluent end of the exposure chambers. Labeled polyethylene tri-pour beakers were rinsed using water from chambers C then B at each test material concentration to attempt to saturate binding sites that could interact with the test material. Chamber A was sampled in all cases by removing 250-500 mL of overlying water from the exposure chamber.
- Sample storage conditions before analysis: N/A

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stabilized well water was delivered to a constant temperature room (air temperature approximately 20 degrees C) housing the entire test system. Well water was filtered through a 0.2 micro meter pore filter prior to entering the dilutor. The dilutor was constructed to deliver 0.5L to each group of replicate chambers. Automatic solid state timers were programmed to deliver water (4 min/cycle) to initial receiving chambers while a peristaltic pump set for a delivery rate of 1.35 mL/min or about 2.70 mL/cycle, delivered a set volume of toxicant to a mixing chamber. Toxicant and dilution water were automatically mixed in a serial fashion, dilution factor of 0.5 resulting in 100%, 50%, 25 %, 12.5%, and 6.25% of a set target exposure plus a control.
- Eluate: N/A
- Differential loading: N/A
- Controls: Yes
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc): N/A

Stock solutions were prepared every other day. The microcosms received nominal doses of 0 (control), 12.5, 25, 50, 100, and 200 micrograms/L. Total ADEAC cationic surfactant concentrations measured during the study were relatively consistent and approximated nominal target exposure concentrations. Mean ± SD measured concentrations were 13.7 ± 4.4, 21.5 ± 46.5, 46.5 ± 15.1, 107.1 ± 34.5 and 211.7 ± 33.7 microgram/L for 12.5, 25, 50, 100, and 200 micrograms/L nominal concentrations, respectively. The control measured level of 2.3 ± 2.7 micrograms/L is below the probable limit of quantitation.

Test organisms

Test organisms (species):

other: Natural periphyton communities

Details on test organisms:

TEST ORGANISM
- Common name: Natural periphyton communities
- Strain: N/A
- Source (laboratory, culture collection): Natural periphyton communities were obtained by colonizing artificial substrates in the upper reach of the Little Miami River, north of Xenia, Ohio in Giles County. The site chosen for colonization was a reach of high quality which has been previously characterized by Lewis (1986), Lewis et al. (1986) and Wynes and Wissing (1981). The river is dominated by a diverse diatom assemblage, is well buffered (total hardness approximately 250 mg/L, total alkalinity approximately 200 mg/L), and is slightly alkaline (pH values above 8.2 and greater are common). Substrate holders were placed in the river on December 15, 1994 above the Xenia waste water treatment plant outfall. The location was devoid of canopy at the time of the study. Water depth ranged from 0.2 to 0.5 meters deep.
- Age of inoculum (at test initiation): N/A
- Method of cultivation: The experiment reported here used unglazed terra cotta clay tile (2.5 x 2.5 cm). Tiles were fixed in a matrix of 3 x 6 on a Plexiglas plate and secured to cement blocks. Cement blocks were 61 cm long x 31 cm wide. The front of the block was pitched at a 25 degree leading up to the Plexiglas plate which was then recessed below the top lip of the cement so that eddies on the tiles were minimized. Eighteen cement blocks holding a total of 324 tiles were placed in the river. Cement blocks have been left in this location since June, 1990. When tiles are retrieved for conducting a test, new tiles on Plexiglas plates are put in place to initiate colonization for subsequent experiments.

Tiles from the Little Miami River were collected on February 14, 1995 and transported in a cooler with Little Miami River water to Cincinnati and transferred to dilutor exposure chambers. River conditions were clear, water temperature was approximately 4 degrees C, and tiles were found at a depth of 15.2 cm. Five tiles were placed in each replicate chamber resulting in a total of 15 tiles distributed across three replicates at each concentration.


ACCLIMATION
- Acclimation period: None. The substrates were removed from the Little Miami River, and 3 hours later were in the lab test of the test material.
- Culturing media and conditions (same as test or not): N/A
- Any deformed or abnormal cells observed: N/A

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Post exposure observation period:

N/A139

Test conditions

Hardness:

139-163 mg/L as CaCO3

Test temperature:

13.2-16.1 degrees C
air temperature approximately 20 degrees C, Test Temperature was maintained at 10-15 degrees C during this winter-time study which was approximately 5-10 degrees C greater than the ambient colonization conditions.

pH:

8.19-8.52

Dissolved oxygen:

10.4-14.3 mg/L

Salinity:

N/A

Nominal and measured concentrations:

The microcosms received nominal doses of 12.5, 25, 50, 100, and 200 micrograms/L.
Mean ± SD measured concentrations were 13.7 ± 4.4, 21.5 ± 46.5, 46.5 ± 15.1, 107.1 ± 34.5 and 211.7 ± 33.7 micrograms/L , respectively.
The mean measured concentrations were 86-110% of nominal. Details of the analytical results are provided in Table 1 below.

Details on test conditions:

TEST SYSTEM
- Test vessel: The exposure system was a Mount and Brungs (1967) type proportional dilutor. The dilutor was constructed to deliver 0.5L to each group of replicate chambers and was constructed of single strength window glass glued together with silicone sealant. Automatic solid state timers were programmed to deliver water to initial receiving chambers while a peristaltic pump set to deliver the toxicant to a mixing chamber. At the set rates the hydraulic residence time of exposure chambers was approximately 1.5 hr and was confirmed to be 94 ± 17 min. Toxicant and dilution water were automatically mixed in a serial fashion, dilution factor of 0.5 resulting in 100%, 50%, 25 %, 12.5%, and 6.25% of a set target exposure plus a control, and delivered to 6 distribution boxes. Distribution boxes split the flow to three replicate exposure chambers for a total of 18 exposure chambers. Exposure chambers were 325 mm long x 128 mm wide with a water depth of 10.2 mm. Tubing within the dilutor system was silicone rubber with an inner diameter or 47 mm.
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: N/A
- Aeration: N/A
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): 1.5 hours
- Initial cells density: N/A
- Control end cells density: N/A
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): N/A


GROWTH MEDIUM
- Standard medium used: N/A
- Detailed composition if non-standard medium was used: N/A


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Stabilized well water, filtered through a 0.2 um pore filter prior to use.
- Total organic carbon: N/A
- Particulate matter: N/A
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity: N/A
- Ca/mg ratio: N/A
- Conductivity: N/A
- Culture medium different from test medium: N/A
- Intervals of water quality measurement: N/A


OTHER TEST CONDITIONS
- Sterile test conditions: N/A
- Adjustment of pH: N/A
- Photoperiod: Lighting was maintained on a light:dark cycle of 15 hours:9 hours.
- Light intensity and quality: Grow Lux light bulbs were suspended 47 cm above the exposure chambers and light intensity was 530 ± 60 uE/m2/s.
- Salinity (for marine algae): N/A


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Algal scrapings were prepared for in depth taxonomic analysis to quantify effects of alkyl dimethyl ethanol ammonium chloride on periphyton. On week 0 five randomly selected tiles were scraped prior to placement in exposure chambers. Tiles were subsequently sampled from the chambers on weeks 1, 2, 3, and 4. The position chosen for removal was used for all exposure chambers on the same sampling day. Tiles were removed one at a time from each chamber, scraped clean with a razor blade and preserved using 3 % glutaraldehyde. Samples were brought up to a constant volume (100 mL) and sent to the contractor in 250 mL plastic bottles. Algal taxonomy used procedures described in Belanger et al. (1994). Subsamples were initially scanned at 400X and lOOOX for the counter to become familiar with the flora. A 0.1 mL subsample was pipetted into a Palmer-Maloney nannoplankton counting chamber and algae were enumerated and identified using random fields and transects (dependent on the silt content and sample density). The first 500 algal units were recorded. Non-diatom taxa were identified to genus or species when possible. Diatom taxa were identified to lowest practical taxon (usually centric, pennate, cymbelloid). Samples were acid-cleaned and diatom species were subsequently identified at lOOOX magnification using oil immersion light microscopy. Early in the study green algal filaments were not very abundant. By the fourth week, green algae appeared as visual dominant. Because these filamentous algae are difficult to count by Palmer-Maloney counting techniques due to contagious clumping and distribution a strategy to account for these taxa was devised. For week 4 samples, the algae were homogenized in a blender and the previously described counting technique was used to quantitate total cell density. Counts from weeks 0 to 3 were primarily unicellular algae and, therefore, these samples were not homogenized. Taxon specific proportions were constructed from initial Palmer-Maloney chamber counts and applied to total cell densities following homogenization. Based on the identifications, absolute and proportional abundance based on cell number and biovolume were calculated.
- Chlorophyll measurement: N/A
- Other: Samples to determine water quality characteristics were taken weekly for pH, temperature, dissolved oxygen, total hardness, total alkalinity and conductivity.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study
- Test concentrations: N/A
- Results used to determine the conditions for the definitive study: N/A

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

> 212 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Taxa richness

Remarks on result:

other: Trend downward relative to control

Effect concentrations 2

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

107 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Taxa diversity (cell density)

Remarks on result:

other: Trend downward relatve to control

Effect concentrations 3

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

107 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Taxa diversity (biovolume density)

Remarks on result:

other: Trend downward relative to control

Effect concentrations 4

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

> 212 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Total cell density

Remarks on result:

other: Trend upward relative to control

Effect concentrations 5

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

> 212 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Total biovolume density

Remarks on result:

other: Trend mixed relative to control

Effect concentrations 6

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

107 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Dissolved Oxygen (DO)

Remarks on result:

other: Trend downward relative to control

Effect concentrations 7

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

22 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Melosira varians (a dominant taxa)

Remarks on result:

other: Trend downward relative to control

Effect concentrations 8

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

107 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Fragilaria capucina v. mesolepta (a dominant taxa)

Remarks on result:

other: Trend downward relative to control

Effect concentrations 9

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

22 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Gomphonema olivaceum (a dominant taxa)

Remarks on result:

other: Trend upward relative to control

Effect concentrations 10

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

> 212 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: 7 of 10 dominant taxa

Remarks on result:

other: No trend relatve to control

Details on results:

- Exponential growth in the control (for algal test): N/A
- Observation of abnormalities (for algal test): N/A
- Unusual cell shape: N/A
- Colour differences:
- Flocculation: N/A
- Adherence to test vessels: N/A
- Aggregation of algal cells: N/A
- Other: N/A
- Any stimulation of growth found in any treatment: Yes
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: N/A
- Effect concentrations exceeding solubility of substance in test medium: Yes

Results with reference substance (positive control):

N/A
- Results with reference substance valid?: N/A
- EC50: N/A
- Other: N/A

Reported statistics and error estimates:

Analysis of variance was the principal statistical technique employed to detect population and community responses. Population level data were ln (n+ 1) transformed prior to analysis of variance. Significance was inferred at alpha=0.05 followed by a identification of significantly different groups using Dunnett's Test. Two-tailed tests were performed for community level data and one-tailed tests were used for population level data at alpha = 0.05. Analyses were performed using SAS for Windows on the PC.

Any other information on results incl. tables

Community Structure:

A total of 58 algal taxa were identified during the study. Taxa richness varied from a mean of 15.4 taxa per sample in the initial communities to a high of 25.0 taxa per sample in week 4 control communities. However, richness was never significantly affected at any time by ADEAC. Taxonomic richness of periphyton in this study is equivalent to that of previous studies conducted during summer and fall. Shannon-Weaver diversity was determined based on both cell and biovolume density. A trend towards lower diversity was seen with both metrics indicating underlying shifts in algal population abundances. By the conclusion of the study both cell and biovolume densities were significantly reduced at 212 micrograms/L compared to the control resulting in a no-observed-effect concentration of 107 micrograms/L.

Because communities developed in the field when brought into the laboratory are already mature great levels of community growth are not very likely, especially at the temperatures employed in this experiment. Yet, communities did experience approximately a 2-fold increase in density as indicated by cell and biovolume counts. Total cell and biovolume densities appeared suppressed in the presence of ADEAC by week 3. The 212 micrograms/L treatment was significantly lower than the control by this time. However, between week 3 and 4 these differences were alleviated as growth of selected populations in the surfactant exposures increased while 0 and 14 micrograms/L treatments remained constant in density relative to week 3. Therefore, by the conclusion of the experiment there were no exposure dependent differences for either cell or biovolume density.

Visual observations made during the study suggested several gross taxonomic changes had occurred in ADEAC treatments compared to the control and low ADEAC concentrations. The control treatment especially appeared to have a substantially greater amount of growth on periphyton exposure chamber surfaces than tiles. This high growth was primarily composed

of diatoms as indicated by a brownish coloration as opposed to a grass green coloration found in the higher ADEAC treatments. These qualitative observations led to an attempt to evaluate the chamber periphyton that was not sampled for taxonomic assessments by measuring its dry weight. However, the dry weight measurements were difficult with many problems. The results were not considered to be reliable, and will not be included in this robust summary.

Community Function:

Evolution of dissolved oxygen (DO) by periphyton communities was used as an indication of net community metabolism. The DO displayed a complex profile. Early in the experiment on day 3 numerous significant differences were observed. By the end of the observation period the DO measured in control chambers was less than treatments of 22-212 micrograms/L. From Day 13 to 20 of exposure DO in periphyton exposure chamber were fairly constant at each time period.  On day 27 treatments were frequently significantly different than control.  By the conclusion of the measurement period of 8 hours lighting, the 47 and-212 micrograms/L exposures had significantly less DO than the control. This observation suggests a measurable reduction in community productivity. Biovolume abundance data obtained from tiles sampled one day later showed no significant differences for algal periphyton biomass at this time; however, dry weight data suggested the control chamber may have greater periphytic biomass overall. Because the dissolved oxygen measurement is a reflection of all the biomass present in the system, including that on tiles and chamber walls, DO production scaled by all chamber biomass present may not show a reduction. Unfortunately, all the measurements of biomass in the system are not in equivalent units making a definitive summary difficult. However, the DO measurement remains an integrative measure of community function and a NOEC of 107 micrograms/L was concluded.

Response of Dominant Taxa:

Individual alga taxa can display a wide variety of direct and indirect responses to chemical exposure. Some organisms may be suppressed or enhanced in growth. Others, which are obligate epiphytes may track the response of the host as their "habitat" increases or decreases without being directly affected by the chemical. These analyses are further complicated by the fact that the ability to detect effects is positively correlated with dominance. Population coefficient of variation typically declines with increasing density. Therefore, only dominant taxa are worthwhile for investigating statistically defined responses. Dominance is an arbitrary definition, but for the purpose of the assessment of ADEAC a taxon is considered dominant if it comprised 5% or more of the average cell or biovolume density for any treatment or any week.

In this study three diatom taxa, Melosira varians, Fragilaria capucina v. mesolepta, Synedra ulna, appeared to be negatively affected by exposure. Melosira increased in abundance in controls throughout the study achieving a final cell and biovolume abundance that was twice that of any ADEAC treatment. Groups exposed to 47 -213 micrograms/L were significantly reduced in abundance relative to the control by week 4 resulting in a NOEC of 22 micrograms/L. Fragilaria was not detected until week 3 of the study. Growth of this taxon was significantly reduced at 213 micrograms/L during weeks 3 and 4 resulting in a NOEC of 107 micrograms/L. Synedra density was reduced in the presence of ADEAC, but the measured variance in the control and treated groups was large enough that differences could not be statistically detected in week 4 samples. Of these three taxa, Melosira was the most abundant. Previous studies have indicated that Melosira is exceptionally sensitive to chemical stress. When these three taxa are considered simultaneously significant differences are detected at week 3 and appear to be ameliorated somewhat at week 4. Although the analysis of variance at week 4 is not significant a trend is still evident and the likelihood that the responses are biologically important is high.

Green algal filaments were visually apparent at the termination of the experiment. As presented in the Materials and Methods, quantification of filaments can be problematic and the population level data substantiate this. Variance is high (often 100% of the mean) in spite of large differences in density. This is due to the inherent contagiousness of encountering filament cells when filaments are found they are almost always in multiples. Indication of exposure dependent effects is lacking, although there is a trend towards increasing green algal abundance with increasing concentration. One diatom taxon, Gomphonema olivaceum, was numerically dominant and increased in abundance with increasing ADEAC exposure. Significantly greater densities were indicated at 47-213 micrograms/L. Whether this can be considered an adverse reaction is open to debate. Gomphonema olivaceum is a broadly distributed taxon and it is possible the taxon’s abundance is associated with modest increases in filamentous green algae.

Table 2. NOEC Values Concluded from the Algal Periphyton Test of KDB 

Parameter	Change Relative to Control (trend)	NOEC (ug/L)
Taxa Richness	down	>212
Taxa Diversity (cell density)	down	107
Taxa Diversity (biovolume density)	down	107
Total Cell Density	up	>212
Total Biovolume Density	mixed	>212
Dissolved Oxygen	down	107
Melosira varians	down	22
Fragilaria capucinav.mesolepta	down	107
Gomphonema olivaceum	up	22
7 of 10 dominant diatom taxa	none	>212

NOEC values are based on mean measured concentrations of test substance.

Applicant's summary and conclusion

Validity criteria fulfilled:

not applicable

Remarks:

There are no validity criteria for this type of test

Conclusions:

The effect of ADEAC (KDB) on algae was evaluated in a laboratory microcosm test of algal periphyton communities in a 28 day, flow-through test. Significant effects on several diatom taxa suggest an adverse NOEC of 22 ug/L for algal periphyton communities (mean measured concentration).

Executive summary:

The effect of alkyl (C12 -14) dimethyl hydroxyethyl ammonium chloride (ADEAC or KDB) on algae was evaluated in a flow-through laboratory microcosm study on algal periphyton communities. Periphyton was colonized on clay tile substrata in the field (Little Miami River, Ohio) in December, 1994, retrieved in February, 1995 and transferred to the laboratory for 28d long-term exposures to the cationic surfactant, KDB.

Each treatment was replicated three times and sampled weekly. The nominal test concentrations were 0, 12.5, 25, 50, 100, and 200 ug/L. Mean measured concentrations were 2.3 (control), 13.7, 21.5, 46.5, 107, and 212 ug/L (86 - 110% of nominal). Communities were exposed to KDB at 13-16 deg C during the 28-d period.

Periphytic communities exposed to KDB had significantly reduced diversity and altered community function as measured by dissolved oxygen production at 212 micrograms/L. Cell and biovolume density and taxa richness were unaffected at any concentration. Two dominant diatom taxa (Melosira varians and Gomphonema olivaceum) had reduced population abundances at 47 micrograms/L (NOEC 22 ug/L). A community level no-observed-adverse-effect concentration (NOEC) of 22 micrograms/L was concluded.