Ceratonia siliqua, ext.

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 October 2017 - 13 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

- Physical appearance: Dark brown to black liquid
- Storage of test material: At room temperature

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- No correction was made for the purity/composition of the substance.
- Method: a stock solution of 10 g/L was prepared by adding 4.9924 g to 500 mL of Milli-RO water. Magnetic stirring for 19 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear dark yellow to orange stock solution (pH 5.4) corresponding to the test concentrations were then added to the test media (16 mL synthetic medium and made up to 250 mL with Milli-RO). Thereafter, 250 mL of activated sludge was added resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
- Controls: test medium without test item, treated in the same way as the test item solutions (blank control)

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: the sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.7 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test conditions

Test temperature:

20 - 22 °C

pH:

7.2-7.5 (before addition of sludge), 7.2-8.2 (after the 3-hour exposure period)

Nominal and measured concentrations:

- Nominal concentrations: 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM:
- Test vessel: all glass open bottles/vessels, fill volume: 500 mL
- Aeration: yes; clean, oil-free compressed air - aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/l at 20°C) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): 3 (highest concentration) and 1 (lower concentrations)
- No. of vessels per control (replicates): 6
- No. of vessels for the reference item (replicates): 1
- Suspended solids concentration in final test solution: 1.5 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water purified by reverse osmosis (Millipore Corp., USA)
- Test medium: adjusted ISO medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature - continuously in a control vessel, pH - at the start and the end of the test period

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED: rate of oxygen consumption at the end of the period

RANGE-FINDING STUDY:
Since the results from the combined limit/range-finding test were adequate for determination of the effect concentrations, no further testing was performed and the combined limit/range-finding test was reported as the final test.

Reference substance (positive control):

yes

Remarks:

3.5-dichlorophenol (3,5-DCP)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

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

Effect concentrations 2

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- No EC50 value could be determined as no effects were observed at the highest tested concentration (the EC50 value exceeded the highest concentration).
- The test conditions during the exposure period were within the requirements prescribed in the study plan.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Tested concentrations: 1.0,3.2, 10 and 32 mg/L
- The EC50 for 3,5-DCP was 6.62 mg/L, 95% CI: 3.20-10.12 mg/L

Reported statistics and error estimates:

ECx calculation:
For the reference item, calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
For the test item, no ECx values could be calculated.

NOEC calculation:
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of the respiration rate (Step-down Jonckheere-Terpstra Test Procedure and Williams Multiple Sequential t-test Procedure for the first and second full test, respectively, with: α=0.05, one-sided, smaller).

Calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH,

Any other information on results incl. tables

Table 1 Results - Respiration Rate/Inhibition and pH Values

Replicate	Concentration (mg/L)	pH		Respiration rate		% Inhibition respiration rate (mean value)
		Start	End	(mg O2/L.h)	(mg O2/g.h)¹
C 1	0	7.5	8.0	33.10	22.07
C 2	0	7.5	7.9	31.42	20.95
C 3	0	7.4	7.9	29.48	19.65
C 4	0	7.4	7.9	29.62	19.75
C 5	0	7.4	7.8	26.99	17.99
C 6	0	7.4	7.9	26.66	17.77
C Mean				29.55	19.70 (RC)
SD				2.49	1.66
CV (%)				8	8
R 1	1.0	7.5	8.1	28.24	18.83	4.42
R 2	3.2	7.5	8.2	17.16	11.44	41.92
R 3	10	7.5	8.2	12.34	8.23	58.23
R 4	32	7.5	8.2	6.49	4.33	78.03
T 1	10	7.4	8.0	25.31	16.87	14.33
T 2	100	7.4	7.8	35.54	23.69	-20.29
T 3a	1000	7.2	7.9	26.57	17.71	10.07
T 3b	1000	7.2	7.9	30.24	20.16	-2.35
T 3c	1000	7.2	7.7	39.75	26.50	-34.54
T3 Mean				32.19	21.46 (RT)	-8.94 (IT)
TA	1000	7.2	7.2	0.00^#	0.00^#	100.00

C:      Control; R:      Reference item, 3,5-dichlorophenol; T:      Test item, Carob Bean Extract; TA:    Abiotic control of Carob Bean Extract; SD:    Standard deviation; CV:   Coefficient of variation

¹         The amount of suspended solids in the final test mixture was 1.5 g/L.; R_C:     Total respiration in the control; R_T:      Total respiration with Carob Bean Extract; I_T:       % inhibition of total respiration relative to R_C

^# No respiration, therefore expressed as 0 mg O₂/L.h (see paragraph 6.1)

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks'

Conclusions:

In a respiration inhibition test, Carob Bean Extract was not toxic to waste water bacteria at and below a concentration of 1000 mg/L (NOEC). The EC50 for inhibition of total respiration exceeded the highest tested concentration (>1000 mg/L).