Trilithium hexafluoroaluminate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date : 31/10/2013, study completion date : 12/05/2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The concentrations of the test substance were determined for the replicates with alga inoculums at the beginning and the end of the test. Before sampling, separation of algae from the test medium was made by syringe filters. For the extra replicates, every 24 hours after the test initiation, samples were taken for chemical analysis.

Vehicle:

no

Details on test solutions:

Range-finding test :
During the pre-test , a white precipitation was observed in the test solution of the treatment groups. The test substance (molecular formula: Li3AlF6) contains aluminum (Al) element, so the precipitation could result from the formation of low soluble salts, i.e., the reaction product between aluminum and anions present in the test water. The test solution of each treatment was prepared at its respective nominal concentration: a certain amount of the test substance was added into the alga medium, and stirred (via magnetic stirrer) for 72h at 500rpm in the dark followed by filtration using a 0.45μm nitrocellulose membrane. After filtration the test solutions were clear and no precipitation was visible.

Definitive test :
The test solution of each treatment was prepared at its respective nominal concentration: a certain amount of the test substance was added into the alga medium, and stirred (via magnetic stirrer) for 72h at 500rpm in the dark followed by filtration using a 0.45μm nitrocellulose membrane. After filtration the test solutions were clear and no precipitation was visible.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test was performed with Pseudokichneriella subcapitata (ID: PS-IHB-130822) which was originally supplied by the institute of hydrobiology, Chinese Academy of Sciences. This common species is one of the species recommended in the OECD TG 201. Ease of culturing and handling, sensitivity to a variety of chemical substances, and the extensive data base for this species make them suitable for acute toxicity testing.

For evaluation of the algal quality and experimental conditions, potassium dichromate was tested as a positive control twice in 2013 to demonstrate satisfactory test conditions. The 72h-ErC50 values of the test substance to Pseudokirchneriella subcapitata were 0.83mg/L (95% CI 0.76-0.89mg/L) and 0.84mg/L (95% CI 0.74-0.92mg/L) , respectively, which were within the defined range of 1.10±0.48mg/L, recommended based on an international ring test(Pattard et al, 2009).

The algae were inoculated in 250mL glass flasks with a concentration of 10E4 cells/mL, incubated during 3 days and then inoculated to another flask. At the beginning of the test, the algae were in the exponential growth phase when inoculated into the test solutions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

No data

Test temperature:

Measured from 22.9 to 23.5°C in the definitive study.

pH:

Measured from 8.15 to 9.52 in the blank control of the definitive study.
Measured from 6.73 to 8.07 in the treatment groups of the definitive study.

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

Range-finding test: three nominal concentrations of 12.0mg/L, 60.0mg/L and 120mg/L were used. The concentrations were not measured.
Definitive study: five nominal concentrations of 3.00mg/L, 5.40mg/L, 9.60mg/L, 16.8mg/L and 30.0mg/L respectively corresponding to geometric mean measured concentrations of fluoride: 2.14mg/L, 3.66mg/L, 6.20mg/L, 10.4mg/L and 17.9mg/L and geometric mean measured concentrations of aluminum: 0.0437mg/L, 0.121mg/L, 0.148mg/L, 0.476mg/L and 1.58mg/L.

Details on test conditions:

Algal medium : Analytical grade salts were dissolved in deionised water to obtain separate alga medium stock solutions. The stock solutions were filtrated with 0.22μm filter membrane and kept at 4°C. The alga medium was prepared by diluting the stock solutions in deionised water shortly before the test began.

Test vessels: 250 mL glass conical flasks

Conditions of exposure:
- Incubation: Each flask was capped with Parafilm and put in a culturing chamber by shaking at a speed of 130rpm.
- Duration: 72h;
- Temperature: 21-24℃;
- Illumination: the surface where the cultures are incubated should receive continuous, uniform fluorescent illumination at equivalent range of 4440–8880 lux for cool white light.

Procedure:
Test solutions: Before the initiation of the test, pH values of the respective test solution were adjusted to approximately 7.0 by 1M HCl. Three replicates were included for each treatment as well as six replicates for the blank control . Because of relatively large sampling volumes , one additional replicate with test solution was prepared for each concentration level for chemical analysis during the test. These replicates were treated identically to those used for testing but no alga inoculum was added.

Inoculation and incubation: An inoculum culture in the test medium was pre-cultured for 3 days before the start of the test under the test conditions. At the beginning of the test, the algae were in the exponential growth phase when inoculated to the test solutions and the density of the alga was 1.730×10E6/mL and 1.945x10E6/mL in the range-finding study and the definitive study respectively. In the range-finding study 1.73 mL of the alga culture and in the definitive study 1.54 mg/L of the alga culture were inoculated into 300mL of test solution for each treatment to obtain an initial cell density of the test solutions of about 10E4 /mL. 100mL of test solution was added into each of 250mL glass conical flasks.

Obervations and measurements: The density of alga cells in each flask was counted and light density in the culturing chamber was determined at 0, 24, 48 and 72 hours.At the beginning of the test, pH values for the control and each treatment were measured using the remaining test solutions. At the end of the test, one vessel was randomly taken from the treatment group and the control group for pH measurement and microscopic observation of the algae. The concentrations of the test substance were determined at the beginning and the end of the test. Before sampling, separation of algae from the test medium was made by syringe filters.

Results of the range-finidng study to define the concentrations in the definitive study:
Mean specific growth rate inhibition at 72h :
40.9% at 12 mg/L
92.9% at 60 mg/L
91.6% at 120 mg/L.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 11.3 - 23.8 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.96 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 CI : 3.95 - 11.4 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.57 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI : 1.46 - 11 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.44 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI : 0.177 - 4.17 mg/L

Details on results:

- Analytical measurements of the test item:
In the definitive test, the measured concentrations of fluoride for all treatment groups varied within ± 20% from their initial ones (-7.00% -14.5%) and the measured concentrations of aluminum also varied within ± 20% (-9.06% -15.1%) except the exposure concentration at 5.40 mg/L.
Since the test substance is a metal compound consisting of several elements (Li, Al and F), the results were calculated using the nominal concentrations in order to reflect the toxicity of the test substance.

- Validity criteria:
- Exponential growth in the control : yes. The mean biomass increase in the control cultures was 116 , which is higher than the validity criterion of at least 16 within the 72-hour test period.
The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 24.9%, which was below 35%.
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.43% which was below 7%.
The test fulfilled the above requirements and was therefore considered to be valid.

At the end of the test, no abnormal alga cells were observed in the control group by microscopic examination, but swollen cells were seen at the concentration of 30.0mg/L.

Results with reference substance (positive control):

For evaluation of the algal quality and experimental conditions, potassium dichromate was tested as a positive control twice in 2013 to demonstrate satisfactory test conditions. The 72h-ErC50 values of the test substance to Pseudokirchneriella subcapitata were 0.83mg/L (95% CI 0.76-0.89mg/L) and 0.84mg/L (95% CI 0.74-0.92mg/L) , respectively, which were within the defined range of 1.10±0.48mg/L, recommended based on an international ring test(Pattard et al, 2009).

Table 1: The measured concentrations offluoride inthe testsolutionsin the definitive test

Group		Measured Concentration (mg/L)		Geometric mean (mg/L)	72h vs.0h (%)
		0h	72h
Blank Control		ND2	ND2	—	—
Treatment Group (Nominal concentration,mg/L)	3.00	2.00	2.29	2.14	14.5
	5.40	3.59	3.74	3.66	4.18
	9.60	6.21	6.20	6.20	-0.16
	16.8	10.4	10.5	10.4	0.96
	30.0	18.6	17.3	17.9	-7.00

LOD2=0.0665mg/L;ND2, not detected;“—”: not applicable

Table 2: The measured concentrations ofalumimun inthe testsolutionsin the definitive test

Group		Measured Concentration (mg/L)		Geometric mean (mg/L)	72h vs.0h (%)
		0h	72h
Blank Control		0.00177	0.00543	—	—
Treatment Group (Nominal concentration,mg/L)	3.00	0.0408	0.0468	0.0437	14.7
	5.40	0.0974	0.151	0.121	54.8
	9.60	0.138	0.159	0.148	15.1
	16.8	0.496	0.457	0.476	-7.87
	30.0	1.66	1.51	1.58	-9.06

“—”: not applicable

 

Table 3: Cell density ofPseudokirchneriella subcapitataexposed to the test substance for 72 hours in the definitive test

Group			Cell density (cell/mL)
			0h	24h	48h	72h
Blank Control		1	10000	69360	302200	1014000
		2	10000	71220	410600	1316000
		3	10000	73730	362200	1207000
		4	10000	71220	326900	1019000
		5	10000	74300	291300	1101000
		6	10000	76200	374000	1290000
		Mean	10000	72672	344533	1157833
		CV (%)	0	3.45	13.3	11.5
Treatment Group (Nominal concentration,mg/L)	3.00	1	10000	73330	337000	894800
		2	10000	68560	304400	844700
		3	10000	72760	378900	946300
		Mean	10000	71550	340100	895267
		CV (%)	0	3.64	11.0	5.67
	5.40	1	10000	76500	285300	795300
		2	10000	64560	211400	776600
		3	10000	71970	345100	794100
		Mean	10000	71010	280600	788667
		CV (%)	0	8.5	23.9	1.33
	9.60	1	10000	68490	235900	436400
		2	10000	71040	269100	428600
		3	10000	71700	274000	446400
		Mean	10000	70410	259667	437133
		CV (%)	0	2.41	7.98	2.04
	16.8	1	10000	40460	113900	151000
		2	10000	38520	78180	95810
		3	10000	27858	80250	116800
		Mean	10000	35613	90777	121203
		CV (%)	0	19.1	22.1	23.0
	30.0	1	10000	11630	13660	14940
		2	10000	11770	12310	16700
		3	10000	11850	13800	15780
		Mean	10000	11750	13257	15807
		CV (%)	0	0.95	6.21	5.57

 

Table 4: Growth of Pseudokirchneriella subcapitata for the blank control cultures in the definitive test

Replicate		Average specific growth rate						Yield
		0-24h		24-48h		48-72h			CV (%) for Section-by-section		0-72h
1		0.0807		0.0613		0.0504		23.8		0.0642		1004000
2		0.0818		0.0730		0.0485		25.4		0.0678		1306000
3		0.0832		0.0663		0.0502		24.8		0.0666		1197000
4		0.0818		0.0635		0.0474		26.8		0.0642		1009000
5		0.0836		0.0569		0.0554		24.2		0.0653		1091000
6		0.0846		0.0663		0.0516		24.4		0.0675		1280000
Mean		0.0826		0.0646		0.0506		24.9		0.0659		1147833
CV (%) for replicates		1.72		7.47		7.91		-		2.43		11.6

“—”: not applicable

 

Table 5: Growth ofPseudokirchneriella subcapitataexposed to the test substance in the definitive test

Treatment Group (Nominal concentration,mg/L)		Average specific growth rate					Yield
		0-24h	24-48h	48-72h	0-72h	72h- Inhibition (%)	0-72h	72h- Inhibition (%)
3.00	1	0.0830	0.0635	0.0407	0.0624	5.31	884800	22.9
	2	0.0802	0.0621	0.0425	0.0616	6.53	834700	27.3
	3	0.0827	0.0688	0.0381	0.0632	4.10	936300	18.4
	Mean	0.0820	0.0648	0.0404	0.0624	5.31	885267	22.9
	CV (%)	1.87	5.45	5.48	1.28	—	5.74	—
5.40	1	0.0848	0.0548	0.0427	0.0608	7.74	785300	31.6
	2	0.0777	0.0494	0.0542	0.0604	8.35	766600	33.2
	3	0.0822	0.0653	0.0347	0.0608	7.74	784100	31.7
	Mean	0.0816	0.0565	0.0439	0.0607	7.94	778667	32.2
	CV (%)	4.40	14.3	22.3	0.38	—	1.34	—
9.60	1	0.0802	0.0515	0.0256	0.0524	20.5	426400	62.9
	2	0.0817	0.0555	0.0194	0.0522	20.8	418600	63.5
	3	0.0821	0.0559	0.0203	0.0528	19.9	436400	62.0
	Mean	0.0813	0.0543	0.0218	0.0525	20.4	427133	62.8
	CV (%)	1.23	4.48	15.37	0.58	—	2.09	—
16.8	1	0.0582	0.0515	0.0256	0.0377	42.8	141000	87.7
	2	0.0562	0.0555	0.0194	0.0314	52.4	85810	92.5
	3	0.0821	0.0559	0.0203	0.0341	48.3	106800	90.7
	Mean	0.0655	0.0543	0.0218	0.0344	47.8	111203	90.3
	CV (%)	22.00	4.5	15.4	9.19	—	25.1	—
30.0	1	0.0063	0.0067	0.0037	0.0056	91.5	4940	99.6
	2	0.0068	0.0019	0.0127	0.0071	89.2	6700	99.4
	3	0.0071	0.0063	0.0056	0.0063	90.4	5780	99.5
	Mean	0.0067	0.0050	0.0073	0.0063	90.4	5807	99.5
	CV (%)	6.03	53.3	65.0	11.9	—	15.2	—

“—”: not applicable

 

 

Validity criteria fulfilled:

yes

Remarks:

OECD TG201 validity criteria fulfilled, see section "details on results"

Conclusions:

The toxicity results of the test substance to Pseudokichneriella subcapitata were the following: 72hErC50 = 16.1 mg/L and 72hErC10 = 7.57 mg/L.

Executive summary:

The growth inhibition effect of Lithium cryolite to Pseudokirchneriella subcapitata was determined in a 72 hours test according to the OECD TG 201 and under GLP.

During the pre-test, a white precipitation was observed in the test solution of the treatment groups. The test substance (molecular formula: Li3AlF6) contains aluminum (Al) element, so the precipitation could result from the formation of low soluble salts, i.e., the reaction product between aluminum and anions present in the test water. The test solution of each treatment was prepared at its respective nominal concentration: a certain amount of the test substance was added into the alga medium, and stirred (via magnetic stirrer) for 72h at 500 rpm in the dark followed by filtration using a 0.45μm nitrocellulose membrane.

Based on the results of the range-finding test, five nominal concentrations of 3.00 mg/L, 5.40 mg/L, 9.60 mg/L, 16.8 mg/L and 30.0mg/L (geometric mean measured concentrations of fluoride: 2.14 mg/L, 3.66 mg/L, 6.20 mg/L, 10.4 mg/L and 17.9 mg/L; those of aluminum: 0.0437 mg/L, 0.121 mg/L, 0.148 mg/L, 0.476 mg/L and 1.58 mg/L) were used in the definitive test. One control group of alga medium was run in addition to the treatment groups. Three replicates were included at each concentration as well as six replicates for the blank control .The test duration was 72h.

The mean biomass increase in the control cultures was 116, which is higher than the validity criterion of at least 16 within the 72-hour test period. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 24.9%, which was below 35%. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.43%, which was below 7%. Hence, the test was considered to be valid.

The measured concentrations of fluoride (F) analysed by ion chromatography (IC) for all treatment groups varied within ±20% from their initial concentrations (-7.00%-14.5%). The measured concentration of aluminum (Al) analysed by ICP-MS also varied within ±20% (-9.06%-15.1%) except the exposure concentration at 5.40 mg/L.

Since the test substance is a metal compound consisting of several elements (Li, Al and F), the results were calculated using the nominal concentrations in order to reflect the toxicity of the test substance. Based on average specific growth rate, the 72h-ErC10 value of the test substance to Pseudokirchneriella subcapitata was 7.57 mg/L (95% confidence limits: 1.46 mg/L–11.0 mg/L), and the 72h-ErC50 was 16.1 mg/L (11.3 mg/L–23.8 mg/L) . Based on yield, the 72h-EyC10 was 2.44 mg/L (0.177 mg/L– 4.17 mg/L), and the 72h-EyC50 was 6.96 mg/L (3.95 mg/L–11.4 mg/L).

Description of key information

A reliable toxicity test is available.

Based on the growth rate inhibition, the 72h ErC50 was determined to be 16.1 mg/L and the 72h ErC10 was determined to be 7.57 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

16.1 mg/L

EC10 or NOEC for freshwater algae:

7.57 mg/L

Additional information

One reliable key study is available for this endpoint. In this study, the growth inhibition effect of Lithium cryolite to Pseudokirchneriella subcapitata was determined in a 72 hours test according to the OECD TG 201 and under GLP.

During the pre-test, a white precipitation was observed in the test solution of the treatment groups. The test substance (molecular formula: Li3AlF6) contains aluminum (Al) element, so the precipitation could result from the formation of low soluble salts, i.e., the reaction product between aluminum and anions present in the test water. The test solution of each treatment was prepared at its respective nominal concentration: a certain amount of the test substance was added into the alga medium, and stirred (via magnetic stirrer) for 72h at 500 rpm in the dark followed by filtration using a 0.45μm nitrocellulose membrane.

Based on the results of the range-finding test, five nominal concentrations of 3.00 mg/L, 5.40 mg/L, 9.60 mg/L, 16.8 mg/L and 30.0mg/L (geometric mean measured concentrations of fluoride: 2.14 mg/L, 3.66 mg/L, 6.20 mg/L, 10.4 mg/L and 17.9 mg/L; those of aluminum: 0.0437 mg/L, 0.121 mg/L, 0.148 mg/L, 0.476 mg/L and 1.58 mg/L) were used in the definitive test. One control group of alga medium was run in addition to the treatment groups. Three replicates were included at each concentration as well as six replicates for the blank control .The test duration was 72h.

The mean biomass increase in the control cultures was 116, which is higher than the validity criterion of at least 16 within the 72-hour test period. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 24.9%, which was below 35%. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.43%, which was below 7%. Hence, the test was considered to be valid.

The measured concentrations of fluoride (F) analysed by ion chromatography (IC) for all treatment groups varied within ±20% from their initial concentrations (-7.00%-14.5%). The measured concentration of aluminum (Al) analysed by ICP-MS also varied within ±20% (-9.06%-15.1%) except the exposure concentration at 5.40 mg/L.

Since the test substance is a metal compound consisting of several elements (Li, Al and F), the results were calculated using the nominal concentrations in order to reflect the toxicity of the test substance. Based on average specific growth rate, the 72h-ErC10 value of the test substance to Pseudokirchneriella subcapitata was 7.57 mg/L (95% confidence limits: 1.46 mg/L–11.0 mg/L), and the 72h-ErC50 was 16.1 mg/L (11.3 mg/L–23.8 mg/L) . Based on yield, the 72h-EyC10 was 2.44 mg/L (0.177 mg/L– 4.17 mg/L), and the 72h-EyC50 was 6.96 mg/L (3.95 mg/L–11.4 mg/L).