Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 November 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols/ Current OECD Guidelines and to GLP standards, with the following exceptions: The stability, characterization and verification of the test substance identity and properties, the determination of the concentrations of the test substance in the test solutions by chemical analysis, and the maintenance of records are the responsibility of the sponsor.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

- Concentrations:
1.0, 10, 100, 1000 and 10 000 mg/l

- Sampling method:
After three hours of exposure, or in the case of the controls, three hours within the test vessel, 300 mL of the sludge mixture were transferred into a BOD bottle containing a tefloncoated magnetic stir bar. The bottle was capped with the BOD bottle oxygen probe to eliminate air space and placed on a magnetic stirring plate. The sludge mixture was stirred at a constant rate and the consumption of oxygen over time was recorded on a strip chart recorder . The test material, control or reference toxicant concentration was written on the strip chart paper. The oxygen readings were recorded for ten minutes or until a linear trace covering a sufficient range of oxygen concentrations was obtained.

- Sample storage conditions before analysis:
not specified in report

Vehicle:

yes

Details on test solutions:

The standard reference toxicant for this type of sludge respiration assessment is 3,5-dichlorophenol (97% pure; Aldrich Chemical, Milwaukee, WI; Lot U09905CZ). A stock solution of the reference toxicant was prepared following the methods outlined in DECD 209(OECD, 1984). One-half gram of3,5-dichlorophenol was dissolved in 10 mL ofa IN NaOH solution which was subsequently diluted to 30 mL with WCC laboratory reagent grade water, placed on a magnetic stirrer and stirred with a teflon-coated stir bar, followed by the additionof 8 mL of IN H2S04 (ACS grade). The solution was then brought to a total volume of 1L with WCC laboratory reagent grade water for a nominal concentration of 500 ppm.

- Method:
The following sequence was followed to initiate the test. The first test vessel was used as the first control in the testing series. Sixteen mL of the synthetic sewage feed, 284 mL of dechlorinated tap water, and 200 mL of the sludge inoculum were added to this vessel. The vessel was immediately placed in the temperature-controlled incubator and aerated. Fifteen minutes later, the reference toxicant series was begun. In a separate test vessel, sixteen mL of the sewage feed was added, followed by 274 mL of dechlorinated water, 10 mL of the reference toxicant stock solution, and 200 mL of the sludge inoculum. As with the first control, the vessel was immediately placed in the temperature-controlled incubator and aerated. This process was repeated at fifteen-minute intervals with 20 mL and 40 mL of reference toxicant solution, respectively. For each reference toxicant addition, the volume of dechlorinated water was adjusted so that the total volume within the test vessel remained at 500 mL. The nominal reference toxicant concentrations thus were 10, 20 and 40 ppm, respectively.
Once the series of three reference toxicant test vessels were started, test vessels containing the test material were prepared. As the test material was considered insoluble, no stock solution was prepared and the material was added directly to each test vessel by way of glass microscope cover slips. The nominal test material concentrations were 1, 10, 100, 1,000, and 10,000 mg/L. Thus for 1 mg/L, 0.0005 g was added to the test vessel; for 10 mg/L, 0.0050 g; for 100 mg/L 0.0500 g; for 1,000 mg/L 0.5000 g; and for 10,000 mg/L 5.000 g.
Each vessel was set up at fifteen-minute intervals. After the last test material vessel was prepared, a vessel with no test material was prepared as a test control replicate.

- Eluate:
The sludge/microbial inoculum was exposed to the test material in 1-L glass flint glass bottles. All bottles and glassware used in testing were cleaned with soap, 10% nitric acid, acetone and wee reagent grade water. Low-pressure oil-free air was delivered to each test vessel using a glass pipet. Air pressure was controlled by brass needle valves. All of the test vessels were held in a Forma Scientific Model 3956 water-jacketed incubator in the dark during the three-hour exposure period. The incubator maintained a constant temperature of 20° C.
To measure the rate of oxygen consumption for the activated sludge after exposure to the test materials a Model 51 Yellow Springs Instruments (YSI) dissolved oxygen meter was used.
The oxygen meter was fitted with a BOD bottle probe, with a standard sensitivity membrane and an external output to a Fisher Recordal (Model 5000) strip chart recorder. The strip chart recorder was set at the lOO-m V range and run at 1 cm per minute. To measure the consumption of oxygen, a sub-sample of the exposed sludge was placed in a clean BOD bottle, the change in oxygen concentration over time was measured with the YSI meter and probe, and the results were recorded on the strip chart recorder.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge with associated aerobic organisms were obtained from the Cottonwood Subdivision Wastewater Treatment Plant located in Franklin, Tennessee. This wastewater treatment plant serves only the residential subdivision and its community pool. One gallon of activated sludge was collected by the plant operator, picked up on the day of collection by WCC personnel and transported to the testing laboratory. Immediately upon arrival, thesludge was aerated with low-pressure, oil-free air. The activated sludge organisms were fed a synthetic sewage feed (see below) at a rate of 50 mL per liter.
Synthetic Sewage Medium (per 1 L of solution)
• 16.0 g Peptone (Type I, Sigma Chemical, Lot #32H0025)
• 11. 0 g Beef Extract (Difco Lot #2900 expiration Dec, 1995)
• 3.0 g Urea (Sigma Chemical, Lot #71H0574)
• 0.70 g NaCI (99%+, Aldrich Chemical, Lot #764714-5)
• 0.40 g CaCl2 -2H20 (Fisher Chemical, Lot #915231)
• 0.20 g MgS04- 7H20 (Sigma Chemical, Lot #108F-0255)
• 2.80 g K2HP04 (Sigma Chemical, Lot #121H0637)

Triplicate 4-mL samples of the mixed sludge were dried at 100° C in a Fisherbrand drying oven, until a constant weight was achieved (Table 2-1 attached). Based on these results the sludge was diluted to produce a dry weight per unit volume concentration of 4 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not applicable.

Hardness:

The dilution water for this test was from the City of Franklin, Tennessee, water supply. The water was softened and dechlorinated prior to use. Dechlorination was confirmed with the DPD colorimetric method (APHA, 1989).

Test temperature:

The test vessels were held in a Forma Scientic Model 3956 water-jacketed incubator in the dark. The incubator maintained a constant temperature of 20 degrees C

pH:

no data supplied

Dissolved oxygen:

The oxygen readings were recorded for ten minutes or until a linear trace covering a sufficient range of oxygen concentrations was obtained.

Salinity:

Fresh water used

Nominal and measured concentrations:

1.0, 10, 100, 1000 and 10 000 mg/l

Details on test conditions:

EFFECT PARAMETERS MEASURED

Only the linear section on the chart paper representing the oxygen consumption was used to
evaluate the results. The chart readings were transformed into mg of O2 per liter consumed
per hour (mg o2/Lhr). To calculate the inhibitory effect of the test material, the respiration
rate is expressed as a percentage of the two control respiration rates for each test material
concentration:

1 – [[2XRs]/[Rcl + Rc2]] x 100

where
Rs = oxygen consumption rate of the inoculum exposed to the test material;
Rcl = oxygen consumption rate, Control 1; and
Rc2 = oxygen consumption rate, Control 2.


In addition to the assessment of the test material, the reference toxicant results were similarly
evaluated. The reference toxicant data were subjected to probit analysis (Litchfield &
Wilcoxon, 1949) to calculate the EC50. The EC50 is the concentration of reference toxicant
that produces a 50% reduction in the oxygen consumption of the inoculum. To assure quality
control, the two control consumption rates need to be within 15 % and the 3-hr ECso of the
reference toxicant (3,5-dichlorophenol) needs to be between 5 and 30 mg/L. Both of these
conditions were met for the test reported herein.
To calculate the no-observable-adverse-effect concentration (NOAEC), a t test was performed
following the guidelines in Sokal & Rolhf (Box 9.7; 1981). The test is a comparison between
a single observation and the mean of a sample. The equation for this test is:

ts = [[Y1- Mean]/S2XSQR(N2 + (1/n2))]

with: N2 -1 degrees of freesom
where: ts = the test statistic
Y1 = the test material oxygen consumption rate
S2 = the standard deviation for the mean control oxygen consumption rate
N2 = the number of control observations


Consumption data were evaluated following the guidelines of USEPA(1989) in order to estimate the concentration which would produce a 10% inhibition of oxygen consumption.
The estimate is produced using a linear interpolation and requires that the inhibition increases monotonically witli increasing exposure concentration. If this condition is not met, the data are smoothed to satisfy the condition (USEPA, 1989). The interpolation is:

IC10 = C1 =[{Rc x 0.90) - Rs1] X [(c2-C1)/(Rs2 - Rs1)]}

where: C1 = test concentration at which the inhibition of oxygen consumption is less than 10%
C2 = test concentration at which the inhibition of oxygen consumption exceeeds 10%
Rc = control oxygen consumption
Rs1 = oxygen consumption of microbes exposed to concentration C1
Rs2 = oxygen consumption of microbes exposed to concentration C2

Because the test concentrations were not replicated, the Jackknife statistical method was used to calculated a variance and the 95% confidence interval for the IClO (Sokal & Rohlf, 1981; Box 18.4). This method consists of calculating an ICw using the first control value, then the second control value and finally the mean of the two control values. Additionally, each test concentration is dropped from the calculation in turn. This results in a number of IClO estimates, The variance in these resultant IClO estimates is used to produce a confidence interval with the degrees of freedom based on the number of estimates. This analysis is attached in Appendix E.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: not specified

Duration:

3 h

Dose descriptor:

other: NOAEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: not specified

Duration:

3 h

Dose descriptor:

IC10

Effect conc.:

3 451.4 other: ppm

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 3275.4 to 3627.5 ppm

Details on results:

CONTROL RESPIRATION RESULTS

The raw data collected for the control respiration results are attached in Appendix C. The first control vessel showed a change of2.8 mg/L for oxygen concentration for a 3 5/6-minute period. This equates to an oxygen consumption rate of 43.9 mg O2/Lhr. The second control vessel showed a change in oxygen concentration of 5.3 mg/L for 7 minutes. The second control thus had an oxygen consumption rate of 45.4 mg O2/Lhr. The average for the two controls was 44.7 mg O2/L*hr.

REFERENCE TOXICITY RESULTS
Test inoculum was exposed to three concentrations of 3,5-dichlorophenol: 40, 20, and 10 ppm. The 40-ppm test concentration showed a change of 0.70 mg O2/L for 6 minutes, or a respiration rate of 7.0 mg O2/L hr. The sludge exposed to 20 ppm of the reference toxicant produced an oxygen consumption of 0.7 mg O2/L over a 4-minute period with the respiration rate being equal to 10.5 mg 02/L*hr. The l0-ppm test inoculum consumed 1.3 mg O2/L over a period of 3 116 minutes with a respiration rate equal to 24.6 mg O2/L hr. The resultant consumption rates for the inoculum exposed to the reference toxicant expressed as a percentage of the control were used to calculate the EC50 of the reference toxicant in the sludge used to test the experimental material. Expressed as a percent inhibition compared to the control: the microbes in the 40-ppm test were 84.3 % inhibited: the microbes in the 20-ppm test were 76.5% inhibited; and the microbes in the l0-ppm test were 44.9% inhibited. Plotting percent effect (% inhibition) as a probit value and concentration as a log (base 10) value, the ECso was calculated to be 10.8 mg 3,5-dichlorophenol per liter. The analysis for the EC50 is attached in Appendix D.

TEST MATERIAL RESULTS
Five nominal concentrations of the test material were tested with the activated sludge inoculum; 1, 10, 100, 1,000, and 10,000 mg/L. The results observed during the test are attached in table 3-1 attached.


The results produced in this assessment of the test material did reveal a significant adverse effect of the test material on the oxygen consumption of the microbes associated with activated sludge. The EC50 for this material regarding the inhibition of metabolism, as represented by respiration, is estimated to be greater then 10,000 mg/L. The NOAEC was calculated to be equal to 1,000 mg/L. The IC10 was calculated to equal 3451.4 ppm with the 95% confidence interval from 3275.4 to 3627.5 ppm. The test is limited in that it represents a screening test of the potential effect of the test material on microbial metabolism.

Results with reference substance (positive control):

- Results with reference substance valid?
Yes.

- Relevant effect levels:
The reference material gave a 3-Hour EC50 value of 10.8 mg/l

- Other:
None.

Reported statistics and error estimates:

STATISTICAL ANALYSIS OF THE RESULTS
The resultant respiration rates were respectively; 48.9, 50.0, 50.5, 48.0, and 32.0 mg 02/Lhr. These data translate into percent inhibitions of -9.5%, -12.0%, -13.1 %, -7.5%, and 28.3%. Because none of the test concentration produced an inhibition greater than 50%, an EC50 could not be calculated. The best estimate for the EC50 is that it exceeds 10,000 mg/L. Each respiration rate was statistically compared as a single value versus the mean of the controls following the procedures of Sokal & Rohlf (1981) using a single-sided t test. The results of the t test revealed that the 10,000 ppm test concentration expressed a statistically significant inhibition of oxygen consumption as compared to the controls (Table 3-1 attached). The discriminatory power of the test is low, however, due to having only one degree of freedom. Thus the NOAEC was 1,000 ppm.

An IC10 was calculated using a point estimate technique and Jackknifed to produce a variance from which a confidence could be derived (see Appendix E attached). The result estimate for theICtO was 3,451.4 ppm of the test material. The 95% confidence interval for this estimate is from 3,275.4 to 3,627.5 ppm.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of greater than 10000 mg/l. The No Observed Effect Concentration (NOAEC) after 3 hours exposure was 1000 mg/l.

Executive summary:

The potential impact of the test material on microbial metabolism, as represented by the consumption of oxygen, was investigated using the "Activated Sludge, Respiration Inhibition Test" as prescribed by OECD (1984) and detailed in WCC Protocol OECD 209 (Expanded Range Procedures-See Appendix A attached). The test was performed on November 30, 1993. The test-duration was a three-hour exposure period to the test material followed by up to ten minutes for the measurement of oxygen consumption. Methods The study design was comprised of five nominal exposure concentrations: 1, 10, 100, 1,000 and 10,000 ppm; a duplicate control group; and an assessment of the sensitivity of the inoculum used in the test to a reference toxicant (3,5-dichlorophenol) . The activated sludge respiration test with the test material passed the quality control criteria for an acceptable test. The EC50 calculated for the reference toxicant was 10.8 mg/L, within the acceptable range of 5 to 30 mg/L. The two control replicates produced oxygen consumption rates within the required 15% of each other, 43.9 and 45.4 mg O2/L*hr. The respiration rates of the sludge-associated microbes exposed to the five nominal concentrations of the test material were 48.9, 50.0, 50.5, 48.0 and 32.0 mg O2/L*hr respectively. Results The calculated no-observed-adverse-effect concentration (NOAEC) based on inhibition compared to the control was calculated to be equal to 1,000 ppm. The EC50 was calculated to be greater than 10,000 mg/L. The IC10 was calculated to equal 3451.4 ppm with the 95% confidence interval from 3275.4 to 3627.5 ppm.

Description of key information

The EC50 was calculated to be greater than 10,000 mg/L (OECD 209)

Key value for chemical safety assessment

EC50 for microorganisms:

10 000 mg/L

EC10 or NOEC for microorganisms:

3 451.4 mg/L

Additional information

The potential impact of the test material on microbial metabolism, as represented by the consumption of oxygen, was investigated using the "Activated Sludge, Respiration Inhibition Test". The respiration rates of the sludge-associated microbes exposed to the five nominal concentrations of the test material were 48.9, 50.0, 50.5, 48.0 and 32.0 mg O2/L*hr respectively. The calculated no-observed-adverse-effect concentration (NOAEC) based on inhibition compared to the control was calculated to be equal to 1,000 ppm. The EC50 was calculated to be greater than 10,000 mg/L. The IC10 was calculated to equal 3451.4 ppm with the 95% confidence interval from 3275.4 to 3627.5 ppm. Therefore, the substance can be considered not toxic to aquatic microorganisms in SWTPs.