Reaction product of D-Glucopyranoside, methyl; esterified with oleic acid, methyl ester

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:

2015-03-31 - 2015-04-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Analytical monitoring:

no

Details on sampling:

A mixed population of activated sewage sludge micro-organisms was obtained on 01 April 2015 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

Vehicle:

no

Details on test solutions:

Reference Item Preparation
A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 15 minutes. The pH of this stock solution was measured to be pH 4.4 and was adjusted to pH 7.2 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Preparation of Test System
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. Finally two further control vessels were prepared.
The test was conducted under normal laboratory lighting in a temperature controlled room at approximately 19 °C.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Synthetic Sewage
A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
16 g Peptone
11 g Meat extract
3g Urea
0.7 g NaCl
0.4 g CaCli.2H20
0.2 g MgS04.7H20
2.8 g K1HP04
dissolved in 1 liter of water with the aid of ultrasonication.
The pH of the synthetic sewage stock was 7 .1. The pH value was measured using a Hach HQ160 Flexi handheld meter.

The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C and was used on the day of collection. The pH of the sample was 7.8 measured using a Hach HQ40d Flexi handheld meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (50 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper* using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 °C for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L.
* rinsed three times with 20 mL deionized reverse osmosis water prior to drying in an oven

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

No post exposure observation period reported.

Hardness:

The test water used for the test was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).

Test temperature:

19°C

pH:

7.0 - 8.0

Dissolved oxygen:

7.0 - 8.8 mg O2/L

Salinity:

Not applicable.

Nominal and measured concentrations:

10, 100 and 1000 mg/L nominal

Details on test conditions:

TEST SYSTEM
16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum
- Test vessel: conical flask
- Material, size, headspace, fill volume: 500 mL
- Aeration: with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute.
Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added.
- No. of vessels per concentration (replicates): 1x 10 mg/L und 100 mg/L, 3x 1000 mg/L
- No. of vessels per control (replicates): 4
- No. of vessels per positive control (replicates): 3
- addition of a synthetic sewage as a respiratory substrate


OTHER TEST CONDITIONS
The test was conducted under normal laboratory lighting in a temperature controlled room at approximately 19 °C.
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Observations: Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge.
-pH: The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-Hour incubation period using a Hach HQ40d Flexi handheld meter.
- Oxygen concentration: The oxygen concentrations in all vessels were measured after 30 minutes contact time
- Measurement of the Respiration Rates: As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between 7 mg O2/L and 2 mg O2/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low
oxygen consumption, the rate was determined over an approximate 10 minute period.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.
The relatively large increase in respiration rate observed in some of the test vessels was considered to be due to the possible hormetic response of the activated sewage sludge micro-organisms to the test item, i.e. the stimulation of biological activity due to the presence of test item at concentrations below its toxic level.

Validation Criteria
The coefficient of variation of oxygen uptake in the control vessels was 5.6% and the specific respiration rate of the controls was 20.98 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.

Results with reference substance (positive control):

- Results with reference substance valid
- Relevant effect levels:
EC10 (3h) = 1.0 mg/L
EC50 (3h) = 6.4 mg/L (4.7 - 8.7 mg/L 95% Confidence Limits)

Reported statistics and error estimates:

The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC10, EC20, EC50 and EC50 values determined from the equation for the fitted line.
The EC10, EC20, EC50and EC50 values for the test item were determined by inspection of the inhibition of respiration rate data.
95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data for the range finding test after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001)

Range-Finding Test

The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 1. Oxygen consumption rates and percentage inhibition values for the control, test and reference item after 3 hours contact time are given in Table 2. The pH values of the test preparations at the start and end of the exposure period are given in Table 3. Observations made on the test preparations throughout the range-finding test are given in Table 4. The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L.

No statistically significant toxic effects were shown at all of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Table1: Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mgO2/L)	Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mgO2/L
Control	R1	7.0	79
	R2	7.1	80
	R3	6.4	72
	R4	7.1	80
Test Item	10	6.3	71
	100	6.5	73
	1000 R1	6.8	76
	1000 R2	6.0	67
	1000 R3	6.0	67
3,5-dichlorophenol	3.2	7.5	84
	10	8.2	92
	32	8.8	99

Table 2: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Initial O2Reading (mg O2/L)	Measurement Period (minutes)	Final O2Reading (mg O2/L)	O2Consumption Rates (mg O2/L/hour)	% Inhibition
Control	R1	3.9	3	2.2	34.00	-
	R2	3.9	4	1.9	30.00	-
	R3	5.8	7	2.2	30.86	-
	R4	5.3	6	2.2	31.00	-
Test Item	10	5.0	5	2.2	33.60	[7]
	100	6.7	9	2.3	29.33	7
	1000 R1	3.3	2	2.2	33.00	[5]
	1000 R2	4.0	3	2.1	38.00	[21]
	1000 R3	4.0	3	2.2	36.00	[14]
3,5-dichlorophenol	3.2	6.9	10	3.5	20.40	35
	10	7.4	10	5.3	12.60	60
	32	8.1	10	7.3	4.80	85

Table 3: pH Values of the Test Preparations at the Start and End of the Exposure Period in the Range-Finding Test

Nominal Concentration (mg/L)		PH
		0 Hours	3 Hours
Control	R1	7.6	7.9
	R2	7.7	7.9
	R3	7.8	7.9
	R4	7.7	7.9
Test Item	10	7.7	7.9
	100	7.7	8.0
	1000 R1	7.7	7.7
	1000 R2	7.7	7.7
	1000 R3	7.7	7.6
3,5-dichlorophenol	3.2	7.8	8.2
	10	7.7	8.2
	32	7.8	8.2

Table 4: Observations on the Test Preparations throughout the Test Period in the Range Finding Test

Nominal Concentration (mg/L)		Observations on Test Preparations
		0 Hours*	30 Minutes Contact Time	3 Hours Contact Time
Control	R1	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R2	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R3	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
	R4	Pale yellow/brown dispersion	Dark brown dispersion	Dark brown dispersion
Test Item	10	Pale yellow/ brown slightly cloudy dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	100	Pale yellow/ brown cloudy dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible	Dark brown dispersion, no undissolved test item visible
	1000 R1	Pale yellow/ brown very cloudy dispersion, no undissolved test item visible	Brown dispersion, no undissolved test item visible	Brown dispersion, no undissolved test item visible
	1000 R2	Pale yellow/ brown very cloudy dispersion, no undissolved test item visible	Brown dispersion, no undissolved test item visible	Brown dispersion, no undissolved test item visible
	1000 R3	Pale yellow/ brown very cloudy dispersion, no undissolved test item visible	Brown dispersion, no undissolved test item visible	Brown dispersion, no undissolved test item visible
3,5-dichlorophenol	3.2	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	10	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible
	32	Pale yellow/brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible	Dark brown dispersion, no undissolved reference item visible

Percentage inhibition is plotted against concentration for the reference item, 3,5-dichlorophenol (Figure 1).

Validation Criteria

The coefficient of variation of oxygen uptake in the control vessels was 5.6 % and the specific respiration rate of the controls was 20.98 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.

The validation criterion for the reference item EC50 value was also satisfied

Validity criteria fulfilled:

yes

Remarks:

Scientifically validity criteria were met.

Conclusions:

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

Executive summary:

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)" and in compliance with GLP.

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of approximately 19 °C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC₅₀ value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

The reference item gave a 3-Hour EC₅₀ value of 6.4 mg/L, 95% confidence limits 4.7 to 8.7 mg/L.

Description of key information

A study (Best, 2015) was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)" and in compliance with GLP.

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at a temperature of approximately 19 °C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC₅₀value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

The reference item gave a 3-Hour EC₅₀value of 6.4 mg/L, 95% confidence limits 4.7 to 8.7 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L