Barium chloride

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: US Environmental Protection Agency. 2002. Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms. EPA-821-R-02–012. Washington, DC.

Version / remarks:

2002, fifth edition

Deviations:

yes

Remarks:

minor variations on test water

GLP compliance:

not specified

Remarks:

The test protocol is based on the US EPA (2002) method for C. dubia and is reliable for classification, labelling and risk assessment purposes. The study covers the required exposure duration and includes analytical verification.

Analytical monitoring:

yes

Remarks:

All controls and barium treatments were subsampled at test initiation and termination for analysis of total (unfiltered) and dissolved (<0.45-mm–filtered) multiple metals. Metals were analyzed using inductively coupled plasma– atomic emission spectrometry

Details on sampling:

All controls and barium treatments were subsampled at test initiation and termination

Vehicle:

no

Details on test solutions:

4 different test media have been prepared for this study:

Medium 1: synthetic soft water with sulfate (SSW)
The SSW medium was a modified US Environmental Protection Agency (2002) water (80–90mg CaCO3/L hardness) prepared by adding NaHCO3 (0.096 g/L), MgSO4 7H2O (0.13 g/L), KCl (0.004 g/L), and CaSO4 2H2O (0.06 g/L in a separate beaker) to Milli-Q water to dissolve, with stirring overnight, followed by filtration (0.45mm filter type HA, nitrocellulose membrane; Millipore) and adjustment of pH to 7.2 to 7.5 using 1 M HCl as necessary.

Medium 2: synthetic soft water with chloride instead of sulfate (SSW-Cl)
The SSW-Cl medium was prepared as for SSW except that MgCl26H2O (0.107g/L) was used instead of MgSO47H2O, and CaCl22H2O (0.051g/L) was used instead of CaSO42 H2O.

Medium 3: diluted mineral water that contained low sulfate (DMW)
The DMW media (US Environmental Protection Agency 2002) was prepared by adding 0.2 mL vitamin B12 (100 mg/L) and 0.04mL Na2SeO410H2O (47mg/L) to 2 L of Perrier1 water diluted (20% v/v) with Milli-Q water followed by aeration overnight and a final pH of 7.9 to 8.3.

Medium 4: SSW-Cl supplemented with B12 and Selenium as described for medium 3

--> for the water flea experiment, medium 3 and 4 have been used.


PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Dissolving BaCl2.2 H2O in test media. Barium as BaCl2 .2H2O (Merck) was diluted in glass volumetric flasks or polycarbonate containers with either DMW or SSW- Cl to match the medium of the bioassay. Final test solutions of 1 to 10,000 mg Ba/L were dispensed (20 mL) into duplicate or quadruplicate glass scintillation vials (20 mL). Controls (DMW or SSW-Cl), chloride controls (SSW-Cl), and a copper (CuSO4 .5H O) reference toxicant (DMW) were assessed in parallel with each water flea bioassay for quality control.

- Controls: Chloride controls were used to determine whether the biological response measured when SSW-Cl was used in barium toxicity tests was due to the barium that was added as BaCl2 2H2O or to the additional chloride associated with the barium salt.
- Test concentration separation factor: < 2 in test medium 4 (SSW-Cl + Se and B12). Spacing factor 10 in medium 3 (DMW)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No precipitate was formed in SSW-Cl (medium 4) barium treatments. A white barium sulfate precipitate formed DMW barium treatments and could not be dissolved with acidification. Therefore, total barium in SSW and DMW was based on nominal barium, and only dissolved barium was analyzed.

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

TEST ORGANISM
- Common name: Ceriodaphnia dubia
- Age at study initiation: < 24h. Twenty-four hours prior to test commencement, adults were isolated from the mass cultures, and the resulting neonates (<24 h old) were used in the toxicity tests.
- Source: originally isolated from Lake Parramatta, Sydney, Australia
- Age of parental stock (mean and range, SD):
- Feeding during test: no. Neonates were fed for a minimum of 2h prior to transfer into test solutions without food at test commencement.
- Food type: 400 000 cells/ml Raphidocelis subcapitata (also known as Pseudokirchneriella subcapitata and Selenastrum capricornutum) and 6mg/L of fish food supplement.
- Amount: 5 neonates per replicate.

Test type:

static

Water media type:

freshwater

Remarks:

see details on medium preparation in details on the test solution.

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

80–90 mg CaCO3/L hardness

Test temperature:

25°C
Temperature was monitored throughout testing using temperature recorders with probes in water (MultitripTM Multi Use Data Logger, Temperature; Temprecord International).

pH:

Test medium 4 SSW-Cl + Se and B12: 7.3 - 8.4
Test medium 3 DMW: 7.8 - 8.5
pH was measured using a Thermo Orion Ross combination pH probe (model 815600; Thermo Orion), which was calibrated daily.

Dissolved oxygen:

Test medium 4 SSW-Cl + Se and B12: 89 - 98 % saturation
Test medium 3 DMW: 84-98% saturation
Dissolved oxygen was measured using a WTW Oxi 330 hand- held meter with a CellOx 325 probe and was calibrated daily.

Salinity:

The salinity and conductivity were measured using a hand-held Mettler Toledo Seven Go Duo Pro pH/ion/conductivity meter with an InLab 738-ISM probe. Results not provided in the publication.

Conductivity:

Test medium 4 SSW-Cl + Se and B12: 337 - 14 100 µS/cm
Test medium 3 DMW: 172 - 1750 µS/cm throughout test
The salinity and conductivity were measured using a hand-held Mettler Toledo Seven Go Duo Pro pH/ion/conductivity meter with an InLab 738-ISM probe.

Nominal and measured concentrations:

Test medium 3 DMW:
- Nominal (total Ba): 0, 0.1, 1, 10, 100 mg/L Ba
- Measured dissolved: 0, 0.09, 0.92, 10.23, 92.11 mg/L Ba

Test medium 4 SSW-Cl + Se and B12:
- Total Ba= measured dissolved Ba
Series 1: 0, 0.9, 9.34, 98.36, 981.82, 11336.71 mg/L Ba (2 reps/conc)
Series 2: 0, 2.21, 4.47, 9.68, 14.4, 18.95, 28.93, 51.28 mg/L Ba (2 reps/conc)
Series 3: 0, 4.79, 9.46, 14.06, 18.86, 23.54, 28.47 mg/L Ba (4 reps/conc)

Details on test conditions:

TEST SYSTEM
- Test vessel: 20 mL scintillation vessels
- Material, size, headspace, fill volume: no details in report
- Volume of solution: 20 mL
- Aeration: oxygen saturation was checked, no forced aeration
- Renewal rate of test solution: no renewal, static set-up
- No. of organisms per vessel: 5 neonates per replicate
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates): 4 control replicates
- No. of vessels per vehicle control (replicates): not relevant, no vehicle used

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Barium as BaCl2 .2H2O (Merck) was diluted in glass volumetric flasks or polycarbonate containers with either DMW or SSW- Cl to match the medium of the bioassay

Composition of the media:
- Test medium 3, DMW: 1.6 mg/L Na+, 0.14 mg/L K+, 32 mg/L Ca2+, 0.74 mg/L Mg2+, 4.4 mg/L Cl-, 86 mg/L HCO3-, 5.5 mg/L SO42-
- Test medium 4, SSW-Cl + Se and B12: 26 mg/L Na+, 2.1 mg/L K+, 14 mg/L Ca2+, 13 mg/L Mg2+, 64 mg/L Cl-, 70 mg/L HCO3-, 0 mg/L SO42-
- Intervals of water quality measurement: details on the test water (salinity, conductivity, oxygen, pH, ...) are regularly checked as described above.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH was adjusted to pH 7-8.
- Photoperiod: 16:8-h light:dark cycle
- Light intensity: ambient laboratory light levels (<10 µmol photons m-1 s2)

EFFECT PARAMETERS MEASURED: assessment of mobility of the C. dubia. Immobility was defined as no visible swimming movement after the test solution was gently swirled.

VEHICLE CONTROL PERFORMED: not relevant as no vehicle is used.

RANGE-FINDING STUDY
- Test concentrations: 0, 0.1, 1, 10, 100 mg/L Ba in Test medium 3 DMW and 0, 0.9, 9.34, 98.36, 981.82, 11336.71 mg/L Ba in Test medium 4 SSW-Cl + B12 and Se
- Results used to determine the conditions for the definitive study: see tables provided in the results section.

Reference substance (positive control):

yes

Remarks:

CaSO4.5 H2O The test was acceptable if immobility in DMW controls was <10% and the Cu EC50 was within internal database limits of 6.73.4mg/L (meanSD, n1⁄420).

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

25.78 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

BaCl2

Basis for effect:

mobility

Remarks on result:

other: results in Test medium 4 SSW-Cl + B12 and Se

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

17 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Remarks:

Ba

Basis for effect:

mobility

Remarks on result:

other: results in Test medium 4 SSW-Cl + B12 and Se

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

21.23 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

dissolved BaCl2

Basis for effect:

mobility

Remarks on result:

other: results in Test medium 4 SSW-Cl + B12 and Se

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

14 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

dissolved Ba

Basis for effect:

mobility

Remarks on result:

other: results in Test medium 4 SSW-Cl + B12 and Se

Details on results:

- Abnormal responses: The test was acceptable since immobility in DMW controls was <10%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium: A white barium sulfate precipitate formed in DMW barium treatments, and which did not dissolve with acidification. Therefore, total barium DMW was based on nominal barium, and only dissolved barium was analyzed. No precipitate formed in SSW-Cl barium treatments, and therefore both total and dissolved barium were analyzed.

Reported statistics and error estimates:

Barium effect concentrations that had either a 10, 20, or 50% effect on the biological responses of algal growth inhibition in the case of algae, or immobility in the case of the water flea, were derived using nonlinear regression in the R software package drc (Ritz and Streibig 2005; R Development Core Team 2016).
The ANOVA tests were followed by multiple comparison tests such as Dunnett’s test and equivalent nonparametric statistical tests of significance such as Kruskall–Wallis one- way ANOVA on ranks; these were performed on biological responses across different treatments after testing for normality and homogeneity of variance using NCSS software Ver 07.1.3.

Summary of the effect concentrations in the 2 test media (95% confidence limits in parantheses):

Media type	EC10 (mg/L Ba)		EC20 (mg/L Ba)		EC50 (mg/L Ba)
	dissolved Ba	total Ba	dissolved Ba	total Ba	dissolved Ba	total Ba
Medium 3: DMW	28 (0-240)	29 (0-270)	31(0-260)	33 (0-300)	38 (0-320)	39 (0-370)
Medium 4: SSW-Cl + Se and B12	14 (13-15)	14 (13-15)	15 (14-16)	15 (14-16)	17 (16-18)	17 (16-18)

The similarity of the concentration effect levels for each media type was a result of the steepness of the concentration–response curve between 10 and 20 mg/L barium, suggesting that there was a well-defined toxic threshold in the mechanism of dissolved barium toxicity to water fleas in both media types.

 

Summary - including conversion of Ba to the substance BaCl2:

	SSW-Cl		DMW
effect conc	mg/L Ba	mg/L BaCl2	mg/L Ba	mg/L BaCl2
EC10	14	21.23	28	42.46
EC20	15	22.74	31	47.00
EC50	17	25.78	38	57.62

Results of range finder test in DMW:

Treatment mg/L Ba (total)	48h mobility		standard deviation	n
	%	% of control
0	95	100	11	4
1	90	95	15	2
10	100	105	0	2
100	10	11	15	2
1000	50	53	15	2

Mean 48h water flea mobility in DMW and SSW-Cl controls and chloride controls as a function of nominal Cl concentration and the corresponding Ba-treatment:

Treatment	eq. Ba treatment (mg/L)	Cl (mg/L)	48h mobility (%)	standard deviation	n
DMW control (pre-test)	0	4.4	98	6	12
SSW-Cl control (pre-test)	0	64	97	12	12
DMW control	0	4.4	100	0	12
SSW-Cl control	0	64	93	10	12
SSW-Cl chloride control	10	67	80	0	2
SSW-Cl chloride control	30	72	83	15	6
SSW-Cl chloride control	50	77	100	0	2
SSW-Cl chloride control	1000	320	80	0	2
SSW-Cl chloride control	10000	2600	0	0	2

Preliminary testing comparing 48-h water flea mobility in DMW and SSW-Cl showed mobility of 98 and 97 %, respectively, without a significant difference due to the removal of sulfate from the medium. In subsequent control treatments of barium toxicity tests, water flea mobility was 100% and 93% in DMW and SSW-Cl, respectively, but there was greater variation in the SSW-Cl control response. This increased response variation in SSW-Cl was also evident in the chloride controls conducted in SSW-Cl.

Info on concentrations - dissolved vs total:

There is a high percentage of dissolved barium in DMW and SSW-Cl, i.e. there were no significant differences between the effect concentrations based on dissolved and total measured barium. Although a small percentage (1–14%) of total barium as precipitated barium sulfate was present in the DMW, the water fleas were responding predominantly to the dissolved form of barium in both media type. 

Validity criteria fulfilled:

yes

Conclusions:

The EC10, EC20, and EC50 values based on dissolved barium in SSW-Cl were 14, 15, and 17 mg/L, respectively. The EC10, EC20, and EC50 values based on dissolved barium in DMW were 28, 31, and 38mg/L, respectively. The similarity of the concentration effect levels for each media type was a result of the steepness of the concentration–response curve between 10 and 20 mg/L barium, suggesting that there was a well-defined toxic threshold in the mechanism of dissolved barium toxicity to water fleas in both media types.

Executive summary:

The present study presents the first data on the toxicity of dissolved barium to the water flea C. dubia without confounding effects from precipitated barium sulfate. This was achieved by substitution of sulfate for chloride in the test medium. Modeling of barium speciation demonstrated that the primary factor controlling the proportion of dissolved barium in the test media was sulfate concentration and the formation of barium sulfate. Barium solubility is a crucial factor to consider when toxicity tests and the test media are designed.

In this study Ceriodaphnia dubia were exposed to dissolved barium. The effect of Ba to C. dubia was assessed according to US EPA method (EPA-821-R-02-012). For 48h, C. dubia were exposed to a range of Ba concentrations. Ba was applied in the form of BaCl2.2H2O. The toxicity was assessed in 2 media: 

- Test medium 3 DMW: diluted mineral water

- Test medium 4 SSW-Cl + Se and B12: synthetic soft water with chloride substituted for sulfate. 

The total and dissolved concentration of Ba was assessed throughout the experiment. There was no significant difference between the dissolved and total Ba. In the DMW test medium a bit of precipitation was observed though (1-14%). 

The acute EC10 and EC50 values for 48-h immobilization of the water flea (Ceriodaphnia dubia) by dissolved barium were 14 mg/L (13–15 mg/L 95% CL) and 17 mg/L (16–18 mg/L 95% CL), respectively in test medium SSW-Cl + Se and B12. 

Description of key information

A reliable, measured 48h-EC50 of 17 mg Ba/L (25.78 mg/L BaCl2) has been reported by Golding et al. (2018) , using Ceriodaphnia dubia as test organism. The study has been performed in accordance with EPA-821-R-02-012 and test concentrations have been analytically verified. The result of this study is moreover in line with earlier reported EC50 values, e.g. Biesinger and Christensen (1972) who reported a nominal EC50 of 14.5 mg Ba/L for D. magna.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

17 mg/L

Additional information

The Golding et al. (2018) study presents the first data on the toxicity of dissolved barium to the water flea C. dubia without confounding effects from precipitated barium sulfate. This was achieved by substitution of sulfate for chloride in the test medium. In this study Ceriodaphnia dubia were exposed to dissolved barium. The effect of Ba to C. dubia was assessed according to US EPA method (EPA-821-R-02-012). For 48h, C. dubia were exposed to a range of Ba concentrations. Ba was applied in the form of BaCl2.2H2O. The toxicity was assessed in 2 media: 

- Test medium 3 DMW: diluted mineral water

- Test medium 4 SSW-Cl + Se and B12: synthetic soft water with chloride substituted for sulfate. 

The total and dissolved concentration of Ba was assessed throughout the experiment. There was no significant difference between the dissolved and total Ba. The acute EC10 and EC50 values for 48-h immobilization of the water flea (Ceriodaphnia dubia) by dissolved barium were 14 mg/L Ba (13–15 mg/L 95% CL) and 17 mg/L Ba (16–18 mg/L 95% CL), respectively in test medium SSW-Cl + Se and B12. 

Similar results were obtained in a study by Biesinger and Christensen (1972). The reported 48h-LC50 of 14.5 mg Ba/L for Daphnia magna is based on nominal test concentrations, using the soluble barium chloride dihydrate as test substance. The followed test procedure in this 1972 -study was well described and test conditions (e.g. test medium composition) were properly identified.

No reliable data were identified for the marine compartment