Trilithium hexafluoroaluminate

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 05 September 2013 (Study Plan completion) to 19 December 2013 (Quality Assurance statement)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study performed according to the OECD testing guideline and GLP. However, one of the OECD validity criteria was not fulfilled (i.e. the mean control oxygen uptake rate was 19 mg O2 / g activated sludge in an hour). This deviation was considered to have no adverse effect on the study, therefore it was considered to be valid with restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

Two deviations from this test guideline have been reported but the study integrity was not adversely affected by the deviations: see section “details on results”.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

The batch of Lithium cryolite tested was a white powder with a purity of 95.70%(m). No correction was made for the purity/composition of the test substance.

The test substance was not completely soluble in test medium at the loading rates initially prepared (10, 100 and 1000 mg/l). Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for 24½ hours, except for the abiotic control which was stirred for 26½ hours. Subsequently, 16 ml synthetic medium, 250 ml sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Test system : Micro-organisms in activated sludge.
- Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Number of micro-organisms : Number of micro-organisms was determined as the amount of Mixed Liquor Suspended Solids (MLSS) per litre test medium.
- Preparation of the sludge : The sludge was coarsely sieved (1 mm), washed and diluted with ISO-medium. 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 7.6 on the day of testing. The batch of sludge was used one day after collection; therefore 50 ml of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Medium : Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O : 211.5 mg/l
MgSO4.7H2O : 88.8 mg/l
NaHCO3 : 46.7 mg/l
KCl 4.2 : mg/l

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

None

Hardness:

No data.

Test temperature:

The temperatures measured in the various vessels ranged between 17.6 and 18.4°C and were within the ranges prescribed by the protocol (i.e. 20 +/- 2°C), except for T 1, T 3b and T A (17.1, 17.4 and 23.3°C, respectively)

pH:

The pH in all test series, before addition of sludge was between 7.0 and 7.5. After the 3 hour exposure period the pH was between 6.9 and 8.3.

Dissolved oxygen:

See table 2 in the section "any other information on results".

Salinity:

No data.

Nominal and measured concentrations:

Loading rates: 10, 100 and 1000 mg/L.
The concentrations were not measured.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass open bottles/vessels
- Conditions of exposure: 3 hours during which aeration and stirring took place.
- Material, size, headspace, fill volume: see table 1 in the section "any information on materials"
- Aeration: Clean, oil-free air was supplied. The 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.
- Type of flow-through : not applicable
- Renewal rate of test solution : not applicable
- No. of organisms per vessel: 1.5 g/L of sludge
- No. of vessels per concentration (replicates): The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate.
- No. of vessels per blank control (replicates): 2
- No. of vessels per nitrification control (replicates): 1
- No. of vessels per abiotic control (replicates): 1
- No. of vessels per nitrification control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water: Tap-water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
- Synthetic medium (=sewage feed):
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

PERFORMANCE OF THE TEST AND EFFECT PARAMETERS MEASURED :
The synthetic medium (16 ml) and an appropriate amount of the test substance loading were mixed and made up to 250 ml with Milli-RO water in a 1 litre bottle. The pH was determined. Thereafter 250 ml activated sludge was added. This was the start of the test. The mixture was then aerated during the contact time, using a pipette as an aeration device.
After the 3-hour contact time, the oxygen consumption was recorded for a period of 10 to 12 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. Determination of oxygen was performed with oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.
The pH and temperature were determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations/loading rates and controls.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study and definitive study: a combined limit/range-finding test with loading rates of 10, 100 and 1000 mg/l was performed

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol, CAS RN 591-35-5, Purity 99.7%, source Sigma-Aldrich Chemie GmbH.

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

Remarks:

respiration rate

Details on results:

The combined limit/range-finding test showed 3%, no inhibition and an average of 38% inhibition of the respiration rate at 10, 100 and 1000 mg/l, respectively. The inhibition observed at 1000 mg/l was statistically significant. As discussed with the sponsor no further testing was performed to determine the NOEC. The EC50 was above the highest concentration rate tested (1000 mg/l).

There was no significant oxygen uptake from abiotic processes and the result at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was 21%.

Protocol deviations:
1. The respiration of the control was just below 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour.
Evaluation: This is a deviation of only 1 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour. Furthermore, the response to the reference compound was normal.
2. The temperature of vessel T 1 and T 3b was 17.1 and 17.4°C, respectively. which was lower than the validity criterion of 20 ± 2°C . The temperature of T A was 23.3°C which was higher than the validity criterion.
Evaluation: A small deviation in temperature usually does not influence the results. The responses T 1, T 3b and TA are in the range of what could be expected.
The study integrity was not adversely affected by the deviations.

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/l for total respiration (3.3 mg/l, see table 2).

Reported statistics and error estimates:

EC50:
For the reference substance the percentage inhibition was plotted against the logarithm of the concentrations and the EC50 was determined using linear regression analysis.
For Lithium cryolite no EC50 could be calculated because the EC50 was above the highest loading rate tested.

NOEC estimation:
In the present test Lithium cryolite was slightly toxic to waste water (activated sludge) bacteria at the highest loading rates tested (Two sample t-Test: α=0.05, TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman). As discussed with the sponsor no further testing was performed to determine the NOEC.

Table 2: Study results

Flask	Loading rate (mg/l)	Temp. (°C)	pH before addition of sludge	pH after 3 h contact time	Respiration rate (mg O2/l.h)	Respiration rate (mg O2/g.h)¹	% Inhibition respiration relative to the control (mean value)
C 1	0	18.4	7.5	7.8	32	21
C 2	0	17.9	7.5	7.8	26	17
C Mean					29	19
SD					4	3
CV (%)					15	15
CN	0	18.3	7.5	8.3	14	9	52
R 1	5.0	17.6	7.5	8.1	13	9	55
R 2	12	17.8	7.5	8.2	7	5	76
R 3	30	18.4	7.5	8.2	4	3	86
T 1	10	17.1	7.2	7.9	28	19	3
T 2	100	17.8	7.0	7.7	29	19	0
T 3a	1000	18.2	7.2	7.5	20	13	31
T 3b	1000	17.4	7.2	7.5	16	11	45
T 3c	1000	18.0	7.2	7.5	18	12	38
T 3 Mean		18		12		38*
T A	1000	23.3	7.1	6.9	3	2	90
T N	1000	17.7	7.2	7.7	11	7	212

C: Blank Control

CN: Nitrification control

R: Reference substance, 3,5-dichlorophenol

T: Test substance, Lithium cryolite

SD: Standard deviation

CV: Coefficient of variation

TA: Abiotic control of test substance.

TN: Test substance with N-allylthiourea.

¹ The amount of suspended solids in the final test mixture was 1.5 g/l.

² Relative to CN

* Statistically significant (Two Sample t-Test α=0.05)

Calculations

Calculation of oxygen uptakes

The respiration rate (R) from each vessel, in mg O2/l.h was calculated or interpolated from the linear part of the respiration curve, which was generally between 2 and 7 mg O2/l.

R was calculated by the BlueBox software as (V1 – V2)/Δt * 60

Where:

V1=Value 1, is the oxygen concentration at the beginning of the selected section of the linear phase (mg/l),

V2=Value 2, is the oxygen concentration at the end of the selected section of the linear phase (mg/l),

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

RN = RT - RH

Where:

RN is the rate of oxygen uptake due to nitrification (mg /l.h).

RT is the measured rate of oxygen uptake by the blank- control (no ATU; C) (mg /l.h).

RH is the measured rate of oxygen uptake of the blank- control with added ATU (CN) (mg/l.h).

Furthermore the respiration rate was also expressed as the amount of oxygen consumed per g dry weight of sludge per hour (Rs in mg O2/g.h).

Rs = R / SS

where SS is the concentration of suspended solids in the test mixture (1.5 g/l in the final test mixture).

Calculation of the inhibition

The inhibitory effect (percentage inhibition) at a loading rate was calculated as:

% inhibition = (1 – Rt / mean Rc) * 100 %

in which Rc and Rt are respiration rates of controls and test/reference substance respectively (in mg O2/l.h).

Validity criteria fulfilled:

no

Remarks:

One of the OECD validity criteria was not fulfilled (i.e. the mean control oxygen uptake rate was 19 mg O2 / g activated sludge in an hour). This deviation was considered to have no adverse effect on the study, this study was considered to be valid.

Conclusions:

The EC50 value after 3 hours of incubation was above a loading rate of 1000 mg/l.

Executive summary:

The influence of Lithium cryolite on the respiration rate of activated sludge was investigated after a contact time of 3 hours according to the OECD Testing Guideline No. 209 and GLP.

The test substance was not completely soluble in test medium at the loading rates initially prepared (10, 100 and 1000 mg/l). Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in Milli-RO water with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for 24½ hours, except for the abiotic control which was stirred for 26½ hours. Subsequently, 16 ml synthetic medium, 250 ml sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured by applying continuous aeration and stirring. Thereafter, oxygen consumption was recorded for 10 to 12 minutes.

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/l were tested. The highest loading rate was tested in triplicate, lower concentrations consisted of one replicate. In addition, blank-controls and a nitrification control of the sludge were included. Furthermore, an abiotic control and the highest concentration with a nitrification inhibitor were included.

There was no significant oxygen uptake from abiotic processes and the result at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was 21%.

Of the three validity criteria of the OECD Testing Guideline, only two were fulfilled as described below:

- The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity.

- The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test.

- The mean control oxygen uptake rate was 19 mg oxygen per one gram of activated sludge in an hour.

The deviation of the last criterion was considered to have no adverse effect on the study, this study was considered to be valid.

The combined limit/range-finding test showed 3%, no inhibition and an average of 38% inhibition of the respiration rate at 10, 100 and 1000 mg/l, respectively. The inhibition observed at 1000 mg/l was statistically significant (Two sample t-Test: α=0.05). The EC50 value after 3 hours of incubation was above a loading rate of 1000 mg/l.

Description of key information

A reliable test on the inhibition of the respiration of activated sludge is available.

No NOEC was obtained and the 3h EC50 was determined to be above 1000 mg/L.

Additional information

One reliable key study is available for this endpoint. In this study, the influence of Lithium cryolite on the respiration rate of activated sludge was investigated after a contact time of 3 hours according to the OECD Testing Guideline No. 209 and GLP.

The test substance was not completely soluble in test medium at the loading rates initially prepared (10, 100 and 1000 mg/l). Therefore, 1-Litre test bottles were filled with 200 ml of test substance mixtures in Milli-RO water with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for 24½ hours, except for the abiotic control which was stirred for 26½ hours. Subsequently, 16 ml synthetic medium, 250 ml sludge and Milli-RO water up to 500 ml were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured by applying continuous aeration and stirring. Thereafter, oxygen consumption was recorded for 10 to 12 minutes.

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/l were tested. The highest loading rate was tested in triplicate, lower concentrations consisted of one replicate. In addition, blank-controls and a nitrification control of the sludge were included. Furthermore, an abiotic control and the highest concentration with a nitrification inhibitor were included.

There was no significant oxygen uptake from abiotic processes and the result at 1000 mg/l with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was 21%.

Of the three validity criteria of the OECD Testing Guideline, only two were fulfilled as described below:

- The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity.

- The coefficient of variation of oxygen uptake in control replicates did not exceed 30% at the end of the definitive test.

- The mean control oxygen uptake rate was 19 mg oxygen per one gram of activated sludge in an hour.

The deviation of the last criterion was considered to have no adverse effect on the study, this study was considered to be valid.

The combined limit/range-finding test showed 3%, no inhibition and an average of 38% inhibition of the respiration rate at 10, 100 and 1000 mg/l, respectively. The inhibition observed at 1000 mg/l was statistically significant (Two sample t-Test: α=0.05). The EC50 value after 3 hours of incubation was above a loading rate of 1000 mg/l.