L-arabinose

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:

208-02-21 to 2018-02-21

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))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OCSPP 850.3300: Modified Activated Sludge, Respiration Inhibition Test

Version / remarks:

January 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: AD16081001
- Expiration date of the batch: 2019-08-09

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: Stable under ambient conditions

Analytical monitoring:

no

Details on sampling:

In this study no analytical measurements were performed. Because of the directly added test item the obtained results were referred to the nominal test item concentration.

The oxygen concentration was measured with O2 electrode (working based on LDO method) under stirred conditions and was recorded for about 4 - 10 minutes. The measurement was carried out in completely filled Winkler bottles. For practical reason more O2 electrode were used. Simultaneous (a maximum of four vessels were investigated in parallel) measurements were performed; the test vessels were investigated in 7 cycles with the available four O2 electrodes

The pH and the oxygen concentrations were determined at the start and at the end of the incubation period in the examined test concentration, reference item concentrations and controls. The temperature was measured in the controlled environment room with a min/max thermometer during the incubation period. The water temperature was recorded during the oxygen measurement in all test bottles.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: In the test the test item concentration of 1000 mg/L was examined with five replicates. At the start of the test defined amounts of 5 x 300 mg test item were administered directly into empty containers. The containers were filled up with water and synthetic sewage (to a final volume of 300 mL), just before the inoculation.
Thereafter a pH measurement was carried out and the test item solutions were inoculated. Concentrations in excess of nominal 1000 mg test item/L were not tested.
Because of the test item direct addition an analytical estimation of the test substance concentration in the test vessels was considered as not necessary, and the subsequent calculations referred to the nominal test item concentration.
- Differential loading: No
- Controls: Blank control, abiotic control, reference control with the reference substance 3,5-dichlorophenol, nitrification control
- Evidence of undissolved material: No

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture:
- Name and location of sewage treatment plant where inoculum was collected: Sewage plant for domestic sewage in Balatonfüred, Hungary on 19 February 2018 (two days before the main test).
- Method of cultivation:
- Preparation of inoculum for exposure: The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice.

An aliquot of the final sludge suspension was weighed (7.379 g wet weight), dried and the ratio of wet sludge to dry weight (0.6669 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (27 g dry weight that was equivalent to 299 g wet sludge) was suspended in isotonic saline solution (ad. 9 L) to yield a concentration equivalent to about 3 g per litre (on dry weight basis).
- Pretreatment: The activated sludge was not used on the day of the collection but continuously aerated (2 L/minute) at the test temperature for about 48 hours (2 days) and was fed daily with 50 mL synthetic sewage/L activated sludge. The pH of the activated sludge inoculum was checked after preparation (pH: 7.38), additional pH adjustment of the inoculum was considered not necessary.
- Initial biomass concentration: In the test containers (300 mL final volume) the final concentration of suspended solids, containing 150 mL inoculum was 1.5 g per litre on dry weight basis.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

No

Hardness:

Not specified

Test temperature:

20 +/- 2 °C

pH:

7-8

Dissolved oxygen:

Not applicable

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal limit concentration: 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass flask
- Type: Closed
- Material, size, headspace, fill volume: 300 mL fill volume
- Aeration:
- No. of vessels per concentration: 5 (limit concentration = 1000 mg/L)
- No. of vessels per blank control: 8
- No. of vessels per nitrification control: 3 (with 11.6 mg/L N-allylthiourea)
- No. of vessels per reference control and concentration: 3 (2, 7 and 24.5 mg/L 3,5-dichlorophenol)
- No. of vessels per abiotic control: 3 (with 1000 mg/L of the test item but without inoculum)
- Sludge concentration (weight of dry solids per volume): 1.5 g/L dw
- Nitrification inhibitor used: N-allylthiourea
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Destilled water

Ratio of composition of synthetic sewage referring to 1000 mL:
Peptone 16 g
LAB-LEMCO Powder 11 g
(BEEF extract)
Urea 3 g
NaCl 0.7 g
CaCl2 x 2H2O 0.4 g
MgSO4 x 7H2O 0.2 g
K2HPO4 2.8 g
Distilled water ad. 1000.0 mL


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Complete darkness

EFFECT PARAMETERS MEASURED: Total inhibition of respiration

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable since a limit test was conducted.
- Justification for using fewer concentrations than requested by guideline:
- Range finding study: No. In the frame of this study a preliminary experiment was not performed, but the chosen test item concentration of 1000 mg/L was based on the testing laboratory’s experience with this test item originated from other studies applying bacterial test systems.
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

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

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:

- Any observations that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
- Adsorption: No
- Blank controls oxygen uptake rate: 45.93 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 5.74 %.
- Variation of oxygen uptake rate in control replicates: The observed difference between start and the end blank control values was minimal (~6 %), within the biological variability of the applied system.

Results with reference substance (positive control):

The 3-hour EC50 of 3,5-dichlorophenol was calculated to be 17.7 mg/L, (95 % confidence limits: 16.1 – 19.3 mg/L).

Reported statistics and error estimates:

The 3-hour EC50 value of the reference item and its 95 %-confidence limits were calculated by appropriate statistical test (Probit analysis by IBM® SPSS® Statistics, Version 24 (2016) statistical software program).
Since one concentration (as a limit concentration) of the test item was tested, this concentration level of the test item was compared to the blank control using the 2 Sample t-Test (2-sided, α=0.05) by IBM® SPSS® Statistics, Version 24 (2016) software.

Table 1: The Oxygen Concentrations and pH Values in the Controls and Test Item Treatment Group

Identification	Concentration (mg/L)	Oxygen concentration (mg O2/L)		pH
		Start of the 3-hour aeration	End of the 3-hour aeration	Before the addition of inoculum	Start of the 3-hour aeration	End of the 3-hour aeration
CBA	0.00	7.03	7.41	7.65	7.59	8.60
CBB		7.15	7.34	7.68	7.64	8.48
CBC		7.10	7.14	7.69	7.66	8.58
CBD		6.99	7.28	7.69	7.67	8.54
CBE		7.24	7.30	7.67	7.93	8.52
CBF		7.18	7.41	7.68	7.94	8.48
CBG		7.32	7.46	7.68	7.89	8.44
CBH		7.37	7.08	7.68	7.90	8.46
CNA	11.6 mg ATU/L	7.10	7.50	7.70	7.63	8.53
CNB		7.28	7.27	7.71	7.73	8.60
CNC		7.26	7.50	7.71	7.74	8.48
R1A	2 mg 3,5-DCP/L	7.30	7.54	7.71	7.75	8.58
R1B		7.33	7.18	7.70	7.90	8.57
R1C		7.10	7.26	7.70	7.86	8.53
R2A	7 mg 3,5-DCP/L	7.09	7.46	7.70	7.85	8.43
R2B		7.05	7.26	7.70	7.83	8.54
R2C		7.20	7.27	7.70	7.90	8.46
R3A	24.5 mg 3,5-DCP/L	7.40	7.87	7.69	7.92	8.67
R3B		7.34	7.76	7.68	7.96	8.67
R3C		7.36	7.56	7.68	7.91	8.58
T1A	1000 mg Test Item/L	7.30	7.36	7.70	7.67	8.42
T1B		7.10	7.22	7.70	7.54	8.51
T1C		7.19	7.47	7.70	7.90	8.50
T1D		7.21	7.21	7.69	7.93	8.54
T1E		7.12	7.56	7.70	7.93	8.41
CAA	1000 mg Test Item/L	8.15	8.33	‑ *	7.73	7.42
CAB		8.12	8.05	‑ *	7.73	7.42
CAC		8.16	8.61	‑ *	7.73	7.42

3,5-DCP:  3,5-dichlorophenol

ATU:N-allylthiourea

Remark: *At the Abiotic controls no inoculum was added; therefore one pH measurement was carried outat the start of the test.

Table 2:The Oxygen Consumption Rate (R), Q1, Q2 and the applied Δt valuesin the Controls and Test Item Treatment Group

Identification	Concentration (mg/L)	Oxygen concentration (mg O2/L)		Δt (min)	Oxygen Consumption Rate (R) (mg O2/Lh)	Average R (mg O2/Lh)
		Q1	Q2
CBA	0.00	7.41	2.36	4.5	67.33	68.89
CBB		7.34	2.43	4	73.65
CBC		7.14	2.50	4	69.60
CBD		7.28	2.42	4	72.90
CBE		7.30	2.24	5	60.72
CBF		7.41	2.27	4.5	68.53
CBG		7.46	2.22	4.5	69.87
CBH		7.08	2.51	4	68.55
CNA	11.6 mg ATU/L	7.50	2.26	4.5	69.87	68.89
CNB		7.27	2.16	4.5	68.13
CNC		7.50	2.35	4.5	68.67
R1A	2 mg 3,5-DCP/L	7.54	2.31	4.5	69.73	67.47
R1B		7.18	2.21	4.5	66.27
R1C		7.26	2.28	4.5	66.40
R2A	7 mg 3,5-DCP/L	7.46	2.34	5	61.44	62.11
R2B		7.26	2.23	5	60.36
R2C		7.27	2.43	4.5	64.53
R3A	24.5 mg 3,5-DCP/L	7.87	5.79	10	12.48	13.12
R3B		7.76	5.84	10	11.52
R3C		7.56	5.00	10	15.36
T1A	1000 mg Test Item/L	7.36	2.52	4	72.60	68.60
T1B		7.22	2.21	4.5	66.80
T1C		7.47	2.51	4.5	66.13
T1D		7.21	2.23	4.5	66.40
T1E		7.56	2.23	4.5	71.07
CAA	1000 mg Test Item/L	8.33	8.25	10	0.48	0.50
CAB		8.05	7.94	10	0.66
CAC		8.61	8.55	10	0.36

Q₁: the oxygen concentration at the beginning of the selected section of the linear phase (mg/L);

Q₂: the oxygen concentration at the end of the selected section of the linear phase (mg/L);

Δt: the time interval between these two measurements (min.).

3,5-DCP: 3,5-dichlorophenol

ATU: N-allylthiourea

Table 3: The Specific Respiration Rate (RS)in the Controls and Test ItemTreatment Group

Identification	Concentration (mg/L)	Specific Respiration Rate (mg O2/gh)	Average RS (mg O2/gh)
CBA	0.00	44.89	45.93 CV(%)= 5.74
CBB		49.10
CBC		46.40
CBD		48.60
CBE		40.48
CBF		45.69
CBG		46.58
CBH		45.70
CNA	11.6 mg ATU/L	46.58	45.93   CV(%)= 1.29
CNB		45.42
CNC		45.78
R1A	2 mg 3,5-DCP/L	46.49	44.98   CV(%)= 2.91
R1B		44.18
R1C		44.27
R2A	7 mg 3,5-DCP/L	40.96	41.41   CV(%)= 3.49
R2B		40.24
R2C		43.02
R3A	24.5 mg 3,5-DCP/L	8.32	8.75   CV(%)= 15.23
R3B		7.68
R3C		10.24
T1A	1000 mg Test Item/L	48.40	45.73^n.s.   CV(%)= 4.39
T1B		44.53
T1C		44.09
T1D		44.27
T1E		47.38
CAA	1000 mg Test Item/L	0.32	0.33   CV(%)= 30.20
CAB		0.44
CAC		0.24

^n.s.:Statistically not significant significantly different compared to the control (2 Sample t-Test; α = 0.05)

3,5-DCP: 3,5-dichlorophenol

ATU:N-allylthiourea

Table 4: The Percentage Inhibition (IT) of Total Oxygen Consumption

Identification	Test group	Concentration	Inhibition (IT), of total oxygen consumption (%)
CB (A-H)	Blank Control	‑	0.00
CN (A-C)	Nitrification Control	11.6 mg ATU/L	0.01
R1(A-C)	Reference Item	2 mg 3,5-DCP/L	2.07
R2(A-C)	Reference Item	7 mg 3,5-DCP/L	9.85
R3(A-C)	Reference Item	24.5 mg 3,5-DCP/L	80.96
T1(A-E)	Test Item	1000 mg Test Item/L	0.43
CA(A-C)	Abiotic Controls	1000 mg Test Item/L	99.27

3,5-DCP:3,5-dichlorophenol

ATU:N-allylthiourea

Validity criteria fulfilled:

yes

Conclusions:

In an Activated Sludge Respiration Inhibition Test according to OECD Guideline 209, EU-Method c.11 and US EPA OCSPP 850.3300, the 3-hour EC10, EC50 and EC80 of the test item was determined to be > 1000 mg/L. The NOEC was determined to be 1000 mg/L.

Executive summary:

The toxicity of the test item to aquatic microorganisms was assessed under GLP conditions in an Activated Sludge Respiration Inhibition Test according to OECD Guideline 209, EU-Method C.11 and the Guideline US EPA OCSPP 850.3300. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. Based on these preliminary information the test item L-Arabinose was investigated at the concentration of 1000 mg/L as a limit concentration, only. Defined amounts of the test item were added (measured) directly into the test vessels. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control, nitrification controls and abiotic controls were investigated. The test was performed without pH adjustment. In result, the observed oxygen consumption rates and consequently the specific respiration rates of the test item solutions were in the range of the blank controls. Thus, no inhibitory effect of the test item was observed. The abiotic controls were investigated at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed. The 3-hour EC50 of 3,5-dichlorophenol was calculated to be 17.7 mg/L, (95 % confidence limits: 16.1 – 19.3 mg/L), confirming the suitability of the test system. The specific respiration rate of the blank controls (without the test substance or reference substance) was 45.93 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 5.74 %. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10, EC50 and EC80 values of the test item are higher than 1000 mg/L. The EC50 value was determined as: EC50 > 1000 mg/L. The NOEC was statistically and biologically determined as 1000 mg/L. All validity criteria of the study were met.

Description of key information

In an Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values were determined as greater than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The toxicity of the test item to aquatic microorganisms was assessed under GLP conditions in an Activated Sludge Respiration Inhibition Test according to OECD Guideline 209, EU-Method C.11 and the Guideline US EPA OCSPP 850.3300. The respiration rates (total, heterotrophic and nitrification oxygen uptake rates) of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. Based on these preliminary information the test item L-Arabinose was investigated at the concentration of 1000 mg/L as a limit concentration, only. Defined amounts of the test item were added (measured) directly into the test vessels. In parallel with the test item treatments 3,5-dichlorophenol as positive reference control in a concentrations of 2, 7 and 24.5 mg/L; furthermore blank (inoculum) control, nitrification controls and abiotic controls were investigated. The test was performed without pH adjustment. In result, the observed oxygen consumption rates and consequently the specific respiration rates of the test item solutions were in the range of the blank controls. Thus, no inhibitory effect of the test item was observed. The abiotic controls were investigated at the test item concentration of 1000 mg/L and no remarkable abiotic oxygen consumption was noticed. The 3-hour EC50 of 3,5-dichlorophenol was calculated to be 17.7 mg/L, (95 % confidence limits: 16.1 – 19.3 mg/L), confirming the suitability of the test system. The specific respiration rate of the blank controls (without the test substance or reference substance) was 45.93 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 5.74 %. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10, EC50 and EC80 values of the test item are higher than 1000 mg/L. The EC50 value was determined as: EC50 > 1000 mg/L. The NOEC was statistically and biologically determined as 1000 mg/L. All validity criteria of the study were met.