Phenol, 2-methoxy-, reaction products with 2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane, hydrogenated

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:

From 23 january 2018 to March 2018 (draft report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

No analytical monitoring

Vehicle:

no

Details on test solutions:

The test concentrations were prepared without using a stock solution. The replicates of the test concentrations were prepared by direct addition of the test item to the mixture of deionised water and synthetic sewage feed just before adding the microbial inoculum.

Before starting the exposure (i.e., adding the microbial inoculum) the pH in the test solutions of the definitive test was adjusted to 7.0-7.6 with NaOH where necessary.

A stock solution of 500 mg/L (nominal) was prepared of the reference substance 3,5-dichlorophenol by dissolving 0.5 g 3,5-dichlorophenol in 10 mL of 1N NaOH, diluting to 30 mL with deionised water, and adding 8 mL of 1N H2SO4 under stirring to the point of incipient precipitation. The mixture was diluted to one litre with deionised water. This stock solution was stored in the refrigerator for two months prior to test start. The pH in the stock solution was 7.2.

The various concentrations of the reference item were prepared by diluting the stock solution with deionised water, and subsequently with synthetic feed and inoculum. Respective controls were prepared without the test item.

The total mixture volume for each replicate of the test concentrations and the controls was 500 mL per vessel. Table 1 describes the composition of the test mixtures and the controls. Immediately after addition of the microbial inoculum, the test solutions were aerated. The test vessels were covered with watch-glasses.


Table 1: Composition of test solutions and controls (nominal values)
[1] [2] [3] [4] [5] [6] [7]
code 0.0 A 284 0.0 16 0.0 200
0.0 B 284 0.0 16 0.0 200
C0 0.0 C 284 0.0 16 0.0 200
0.0 D 284 0.0 16 0.0 200
0.0 E 284 0.0 16 0.0 200
0.0 F 284 0.0 16 0.0 200
62.5 A 284 0.0 16 31.25 200
C1 62.5 B 284 0.0 16 31.25 200
62.5 C 284 0.0 16 31.25 200
125 A 284 0.0 16 62.5 200
C2 125 B 284 0.0 16 62.5 200
125 C 284 0.0 16 62.5 200
250 A 284 0.0 16 125 200
C3 250 B 284 0.0 16 125 200
250 C 284 0.0 16 125 200
500 A 284 0.0 16 250 200
C4 500 B 284 0.0 16 250 200
500 C 284 0.0 16 250 200
1000 A 284 0.0 16 500 200
C5 1000 B 284 0.0 16 500 200
1000 C 284 0.0 16 500 200
Ref C1a 1.8 A 282.2 1.8 16 0.9 200
Ref C2a 5.9 A 278.1 5.9 16 2.95 200
Ref C3a 18.8 A 265.2 18.8 16 9.4 200
Ref C4a 60.0 A 224.0 60.0 16 30.0 200
[ 1 ] Nominal concentration in mg/L [ 5 ] Volume of synthetic sewage feed in mL
[ 2 ] Replicate [ 6 ] Nominal amount of of test item in mg per vessel
[ 3 ] Volume of deionised water in mL corresponds to actually applied test item
[ 4 ] Volume of stock solution in mL [ 7 ] Volume of microbial inoculum in mL

Test organisms (species):

activated sludge

Details on inoculum:

Activated sludge from a sewage treatment plant will be used as the microbial inoculum for the test. The activated sludge will be obtained preferably from a sewage treatment works treating predominantly domestic sewage.
The activated sludge will be obtained from the sewage treatment plant and will be washed with reconstituted water on the day of collection. After centrifugation of the sludge the supernatant will be decanted. This washing procedure will be performed at least twice. An aliquot of the washed sludge will be used to determine the dry weight (dw). The result of the weight determination will be used in order to calculate the amount of activated sludge needed for a mixed liquor suspended solids level of 3.75 g dw/L (±10%). This level will result in a final concentration of 1.5 g dw (±10%) mixed liquor suspended solids per litre test medium.
The batch of activated sludge may be stored for a maximum of two days after washing. At the end of each day of storage the sludge will be fed with 50 mL/L of synthetic sewage feed. It will be aerated with clean, oil-free air and kept at a temperature of 20±2 °C.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not applicable

Hardness:

No data

Test temperature:

20.0–20.4 °C

pH:

pH-value of the activated sludge: 6.8
pH value in the test solutions (before addition of the inoculum): 7.2–7.3

Dissolved oxygen:

above 60–70% saturation

Salinity:

Not applicable

Nominal and measured concentrations:

Preliminary non-GLP rangefinding test: nominal concentration levels: 1000, 100 and 10 mg/L
Definitive test: nominal concentration levels: 62.5, 125, 250, 500 and 1000 mg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: glass beakers
- Material, size, headspace, fill volume: filled at 1000 mL glass beaker with watch-glass cover
- Aeration: Yes. The test solutions will be aerated with clean, oil-free air during the test (air flow 0.5–1 L/minute). This is known to keep the dissolved oxygen concentration above 60–70% saturation and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per reference test (replicates): 1
- For each replicate, the test item will be applied once at the beginning of exposure. If the above mentioned preliminary non-GLP range finding test reveals significant inhibition of nitrification (e.g. > 15%) by the test item, three additional replicates of each treatment as well as six replicates for the control, containing 11.6 mg ATU/L, will be used to determine if respiration due to nitrification is inhibited by the test item.
The reference item will be tested at four concentration levels (e.g. 1.8; 5.9; 18.8; 60 mg/L). At least one vessel will be used per treatment. If nitrification is to be determined, additional replicates for each treatment level containing 11.6 mg ATU/L will be used to determine respiration due to nitrification.
- Biomass loading rate: 1500 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water according to OECD Guideline No. 203 will be used to maintain the microbial inoculum before the period of the test. The preparation of reconstituted water is described in ECT’s SOP A2.1.
The final concentration of the salts in the reconstituted water will be:

A) CaCl2 * 2 H2O 294.0 mg/L
B) MgSO4 * 7 H2O 123.0 mg/L
C) NaHCO3 64.8 mg/L
D) KCl 5.75 mg/L

After aeration for at least one hour the physico-chemical characteristics (pH value, temperature, oxygen concentration, conductivity and total hardness) will be determined prior to use.

The required amount of reconstituted water will be prepared within one month before use. The reconstituted water will be aerated during storage.

- Total organic carbon, Particulate matter, Metals, Pesticides, Chlorine, Alkalinity, Ca/mg ratio, Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- Adjustment of pH: Yes
- Photoperiod:Not applicable
- Light intensity: No data

EFFECT PARAMETERS MEASURED : After three hours of incubation (contact time), the content of the first test vessel (control 1) will be poured into the measuring apparatus (consisting of oxygen-electrode, flat bottom flask and stirring device) and the respiration rate will be determined at 2-second intervals over a period of 5–10 minutes or until the oxygen concentration falls below 2 mg/L.
All other test vessels will be measured in the same order that was established for the inoculation of the test vessels (i.e. after a contact time of 3 h each).
Any test vessels containing ATU will be tested in the same way as described above on the same day as the test item.
In case there are indicators of chemical oxygen demand of the test item, the chemical oxygen consumption will be measured by using further test vessels containing test item, synthetic sewage feed and water, but no activated sludge.
The respiration rate (oxygen consumption rate) will be calculated from the oxygen consumption of the test solutions as mg O2/L per hour between 2.0 mg O2/L and 7.0 mg O2/L. The portion of the respiration curve over which the respiration rate will be measured should be linear.
Excursion into O2 concentrations below 2.0 mg O2/L or above 7.0 mg O2/L may be unavoidable and necessary, for example, when respiration is heavily suppressed or if a particular activated sludge respires very quickly. This is acceptable provided the extended sections of the uptake graph are linear and their gradients do not change as they pass through the 2.0 mg O2/L or 7.0 mg O2/L boundaries.
The inhibitory effect of the test item (inhibition of the respiration rate in per cent of the mean control respiration rate) in a particular replicate will be calculated according to the following equation:
[1 – (RS/RC)] x 100 = % inhibition
where

RS = oxygen consumption rate in a selected replicate containing the test item
RC = mean oxygen consumption rate in the six control replicates

In case inhibition of nitrification is to be determined (e.g. as based on the findings in a preliminary non-GLP rangefinder), the inhibitory effect of the test item (inhibition of the rate of nitrification in per cent of the mean control respiration rate) will be calculated after subtracting the oxygen uptake rate in the presence of ATU from the total uptake rate (no ATU present) using the equation above. All calculations of respiration rates and their inhibition will be performed based on the equations given in the test guideline. The equations used will be described in the report.

The oxygen uptake rates measured in the presence of ATU represent heterotrophic respiration only and the differences between these and the corresponding total respiration rates represent nitrification.

The statistical methods to calculate the ECx values will depend on the results of the test. The statistical methods and software used will be recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study: Yes
- Test concentrations: 0, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: The non-GLP range-finding test including a nitrification inhibition control showed less than 50% of control respiration at 1000 mg test item/L. An inhibition of nitrification compared to the control respiration was not found. Since even at 1000 mg/L (highest level to be tested according to the guideline) the overall inhibition of total respiration was less than 50%, a separate evaluation of nitrification was not considered relevant. There was less than 10% inhibition of total respiration at 100 or 10 mg/L. A relevant specific inhibition of nitrification by the test item was not observed in the range-finding test. The definitive test was therefore performed without ATU. Considering the results from the range-finding test, the following nominal concentrations in a geometrical series (spacing factor: 2.0) was tested in the definitive test: 62.5, 125, 250, 500 and 1000 mg test item/L.

Reference substance (positive control):

yes

Remarks:

3-5 dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

318 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

The definitive test showed a weak concentration-response relationship with an inhibition of total respiration rate of less than 20% of control respiration at 1000 mg test item/L.

Results with reference substance (positive control):

The results of this reference test were within the ranges accepted by the test guideline and thereby confirm appropriate test conditions.

Definitive test

Table2: Decrease of total respiration rate (inhibition in % of control) as caused by the test item after 3 h.

 

Treatment [mg test item/L]	Mean respiration rate [mg O2/(l*h)]	Standard deviation	Number of replicates	Inhibition of Respiration rate [% of control value]
Control	62.5	4.12	6
62.5	66.0	7.96	3	-5.6
125	58.8	5.63	3	5.9
250	55.0	6.45	3	12.1
500	51.8	10.77	3	17.1
1000	55.1	6.19	3	11.9

Inhibition (Decrease) [%]: Deviation from the mean of the controls in percent, calculated according to the equation.Negative inhibition represents higher respiration rate than in the controls.

 

Reference test with 3,5 -dichlorophenol:

a) Total Respiration Rate after 3 h exposure:  Respiration rate of activated sludge in (mg O₂/L)/h as dependent on concentration of the test item; Mean: arithmetic mean; Std.Dev.: standard deviation; n: number of replicates; CV: coefficient of variation.

Treatm. [mg/L]	Control	1.8	5.9	18.8	60.0
3 h	62.5^1	64.9	47.8	12.6	5.8
n:	6	1	1	1	1

CV: Coefficient of variation (% of mean value);¹mean control respiration rate

 

b) Respiration Rate in Activated Sludge after 3.0 h.: % Decrease of respiration rate as caused by the test item after 3.0 h.

Treatm.[mg/L]	Mean	Std. Dev.	n	%Decrease
Control	62.51	n.d.	6	0.0
1.8	64.86	n.d.	1	-3.8
5.9	47.76	n.d.	1	23.6
18.8	12.56	n.d.	1	79.9
60.0	5.77	n.d.	1	90.8

Validity criteria fulfilled:

yes

Conclusions:

The inhibitory effect of Rhodiantal original IBCH on the respiration of the activated sludge was investigated in a 3 -hour respiration inhibition test according to OECD guideline for testing of chemicals n°209 (2010).
The following results were determined:
EC50 : > 1000 mg/L (total respiration)
EC10 : 318 mg/L
The validity criteria applied to this study type were met and therefore the study is considered valid.

The coefficient of variation of oxygen consumption rate in control replicates was ≤30%: 6.6%
The oxygen consumption rate in the controls was ≥20 mg O2/(g dw × h): 38.8 mg O2/(g dw × h)
Total respiration: The EC50 (3 hours) of 3,5-dichlorophenol was in the accepted range of 2–25 mg/L: 10.2 mg/L

Executive summary:

The inhibitory effect of Rhodiantal original IBCH on the respiration of the activated sludge was investigated in a 3 -hour respiration inhibition test according to OECD guideline for testing of chemicals n°209 (2010).

Nominal concentrations in the definitive test: 62.5, 125, 250, 500 and 1000 mg test item/L

The following results were determined:

EC50 : > 1000 mg/L (total respiration)

EC10 : 318 mg/L

Based on the results, Rhodiantal original IBCH has no effect on the respiration rate of activated sludge microorganisms.

The validity criteria applied to this study type were met and therefore the study is considered valid.

The coefficient of variation of oxygen consumption rate in control replicates was ≤30%: 6.6%

The oxygen consumption rate in the controls was ≥20 mg O2/(g dw × h): 38.8 mg O2/(g dw × h)

Total respiration: The EC50 (3 hours) of 3,5-dichlorophenol was in the accepted range of 2–25 mg/L: 10.2 mg/L

Description of key information

The 3 -hour EC50 for Rhodiantal original IBCH was > 1000 mg/L.

The 3 -hour EC10 for Rhodiantal original IBCH was 318 mg/L.

Additional information

A GLP-compliant study (P. Egeler, 2018), scored as klimish 1 and flagged as a key study, is available on the Rhodiantal original IBCH.

The following results were determined:

EC50 : > 1000 mg/L (total respiration)

EC10 : 318 mg/L

Based on the results, Rhodiantal original IBCH has no effect on the respiration rate of activated sludge microorganisms.