Coffee, bean, roasted, ext.

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

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 (Carbon and Ammonium Oxidation))

Qualifier:

according to guideline

Guideline:

other: EU Test Method L133

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CF02
- Expiration date of the lot/batch: 31.01.2019
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated in dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: water solubility 2.85 mg/L
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a WAF of the test item. The solutions were stirred for nominal 23 hours and left to settle for approximately 1 hour.
- Final preparation of a solid: not applicable
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: none

FORM AS APPLIED IN THE TEST (if different from that of starting material)
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
OTHER SPECIFICS: none

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF).
- Eluate: not specified
- Differential loading: The range-finding test was conducted at nominal loading rates of 10, 100 and 1000 mg/L. Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 1000, 5000, 10000, 15000 and 20000 mg/L.
- Controls: control water: RO water; positive control: 3,5-dichlorophenol at concentrations of 0.5, 1.0, 5.0, 10 and 25 mg/L.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): cloudy sloution >20000 mg/L.

Test organisms (species):

activated sludge

Details on inoculum:

- Laboratory culture: none
- Name and location of sewage treatment plant where inoculum was collected: Totnes sewage treatment works, Devon , UK
- Method of cultivation: kept aerated at ambient temperature until used in the study
- Preparation of inoculum for exposure: sludge was settled for a short time period and the supernatant decanted
- Pretreatment: none
- Initial biomass concentration: 1500 mg/L

Test type:

other: stirred and aerated

Water media type:

other: RO water

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2°C

pH:

7.5 ± 0.5

Nominal and measured concentrations:

Nominal: 1000, 5000, 10000, 15000, 20000 mg/L
Measured: 929, 4609, 9950, 14270, 16349 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: flask
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 150 mL
- Aeration: yes
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 1
- No. of vessels per abiotic control (replicates): Single replicates of abiotic test substance at 10 and 100 mg/L and triplicate test substance at 1000 mg/L, containing no activated sludge and single blank control replicate.
- Sludge concentration (weight of dry solids per volume): 1500 mg/L
- Weight of dry solids per volume of reaction mixture per unit of time: not specified
- Nutrients provided for bacteria: OECD synthetic sewage feed
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea
- Biomass loading rate: not specified

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: RO water
- Particulate matter: not specified

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to 7.5 ± 0.5 if necessary
- Photoperiod: not applicable
- Light intensity: not applicable
- Details on termination of incubation: not specified

TEST CONCENTRATIONS
- Range finding study: yes, nominal test concentrations of 10, 100 and 1000 mg/L
- Test concentrations: nominal, 1000, 5000, 10000, 15000 and 20000 mg/L
- Results used to determine the conditions for the definitive study: respiration rates, percentage inhibition

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

5 164 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: WAF

Basis for effect:

inhibition of respiration due to nitrification

Results with reference substance (positive control):

The EC50 for total respiration for 3,5-dichlorophenol reference substance was 0.93 mg/L in the range finder test and 4.3 mg/L in the definitive test.
The EC50 for heterotrophic respiration for 3,5-DCP was 14.9 in the range finder test. The EC50 for nitrification respiration for 3,5-DCP was 0.19 in the range finder test.
In the range finder test the 3,5-DCP total respiration EC50 was low, this shows the sludge was more sensitive than expected, however this was deemed to be acceptable as the test substance EC50 was > 1000 mg/L.

Reported statistics and error estimates:

The respiration data obtained at 3 hours was analysed using CETIS. In the definitive test the control was compared to the treatments by two-way analysis of variance and Bonferroni adjusted t-tests to identify significant differences (p < 0.05). The EC50 and 95% confidence intervals were calculated using the linear interpolation (ICPIN) method.

Validity criteria fulfilled:

yes

Conclusions:

In the activated sludge respiration inhibition test, conducted according to OECD test guideline 209 and to GLP, the 3-hour EC50 value for total respiration inhibition was determined at 5164 mg/L measured loading rate as Water Accommodated Fraction.

Executive summary:

A respirometric test conducted according to OECD test guideline 209 and to GLP was performed to determine the effect of Coffee, bean, roasted, ext. on the respiration rate of activated sludge microorganisms.

Activated sludge was obtained from the local Sewage Treatment Works. The settled sludge was fed with 50 mL of OECD synthetic sewage feed per liter of sludge per day and aerated at room temperature, until it was used in the test.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, microorganism were exposed to WAFs of the test item over a range of nominal loading rates of 1000, 5000, 10000, 15000 and 20000 mg/L; or measured loading rates of 929, 4609, 9950, 14270 and 16349 mg/L; 5 replicate flasks per concentration for 3 hours at 20 ± 2°C.

Since no heterotrophic respiration was observed in the range finding test, it was assumed that the total respiration was equivalent to the nitrification respiration, and therefore, only total respiration was measured in the definitive test.

Total Organic Carbon (TOC) analysis of the test preparations was performed using Hach Lange IL500 carbon analyzer.

The 3-hour EC50 value for total respiration inhibition was determined at 5164 mg/L measured loading rate as Water Accommodated Fraction

Description of key information

EC50 (total respiration inhibition): 5164 mg/L

Additional information

A respirometric test conducted according to OECD test guideline 209 and to GLP was performed to determine the effect of Coffee, bean, roasted, ext. on the respiration rate of activated sludge microorganisms.

Activated sludge was obtained from the local Sewage Treatment Works. The settled sludge was fed with 50 mL of OECD synthetic sewage feed per liter of sludge per day and aerated at room temperature, until it was used in the test.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, microorganism were exposed to WAFs of the test item over a range of nominal loading rates of 1000, 5000, 10000, 15000 and 20000 mg/L; or measured loading rates of 929, 4609, 9950, 14270 and 16349 mg/L; 5 replicate flasks per concentration for 3 hours at 20 ± 2°C.

Since no heterotrophic respiration was observed in the range finding test, it was assumed that the total respiration was equivalent to the nitrification respiration, and therefore, only total respiration was measured in the definitive test.

Total Organic Carbon (TOC) analysis of the test preparations was performed using Hach Lange IL500 carbon analyzer.

The 3-hour EC50 value for total respiration inhibition was determined at 5164 mg/L measured loading rate as Water Accommodated Fraction.