Slimes and Sludges, blast furnace and steelmaking

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:

15.1.2010-28.1.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

The study was performed without the analytical determination of the test substance concentrations. Because the test substance is multi-component poorly soluble substance the water-accommodated fractions (WAFs) were prepared for preliminary and full test.

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Since the test substance is multi-component poorly soluble substance the water-accommodated fractions (WAFs) were prepared for preliminary and full test. The WAFs were prepared by adding measured amounts of the test substance to the test medium and then were stirred for suitable period. After period of stirring the WAFs were filtered through 0.45 µm filter and these solutions were used for testing.
The suitable period for achieving equilibrated concentration of dissolved and dispersed components in aqueous phase was determined in preliminary experiment. For preliminary experiment the WAFs were prepared as follows: the suspension of 100 mg of the test substance in 1000 ml of test medium were stirred for period 3, 24, 48 and 72 hours on a shaft stirrer, consequently filtered through 0.45 µm filter. During this experiment the concentrations of metals (Al, Ca, Fe, K, Mg, Mn, Na and Zn) in resulted WAFs were determined. The WAFs were prepared in test medium, which contents metals as Ca, Mg, Na, K etc. For this reason the analytical determination of the concentrations of metals was performed also in pure test medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: Brinkmann 1953/SAG 86.81
- Source (laboratory, culture collection): Botanic Institute of Czech Academy of Science, Trebon
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The strain culture was preinoculated from the stock solution and cultivated in flasks with the test medium on indirect daylight at laboratory temperature. Algae inoculum for the test was sampled from exponentially growing inoculum culture. The strain culture was always set to pre-culturing of cells for 3 days before the start of the test. Inoculum culture was kept 3 days under conditions at which the test was performed. After this period the culture was in the state of the exponential growth and had the cell density suitable for the performance of the test. The cell density of the pre-culture was measured just before the start of the test and the needed inoculum volume was calculated.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.0-22.5°C (preliminary test)
22.0-23.0°C (full test)

pH:

7.7-9.2 (preliminary test)
7.8-8.8 (full test)

Nominal and measured concentrations:

100, 60, 40, 20 and 10 mg/l (nominal concentrations at preliminary test)
100, 83, 69, 58, 48, 40 and 33 mg/l (nominal concentrations at full test)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks with stoppers venting air filled 50 ml of the test solution
- Initial cells density: 10000 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 6000-10000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Burker's counting chamber

Reference substance (positive control):

yes

Remarks:

poassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

58 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

69 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

The inhibition of cell growth in the full test was 48.2 % at the highest test substance concentration 100 mg/l. The inhibition of growth rate in the full test was 14.6 % at the highest test substance concentration 100 mg/l. Therefore exact values of EbL50 and ErL50 could not be calculated and the values of EL are given in the form of a range.
The determination of NOELR values was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 (TriloByte, Czech Republic).
The loading rates of the test substance were used for all evaluations and results.

Results with reference substance (positive control):

The results of the verification test with K2Cr2O7, carried out with the following results:
ErC50 (72 h) = 0.86 mg/l (95% confidence limit: 0.84 – 0.88 mg/l)
EbC50 (72 h) = 0.32 mg/l (95% confidence limit: 0.31 – 0.33 mg/l)
The given results for the ErC50 are convenient to range of the results of the interlaboratory tests. The result of the last interlaboratory test arranged by Centre for Assessment of Laboratories of T.G. Masaryk Water Research Institute Prague in 2008 for potassium dichromate for 72h – ErC50 was:
ErC50 (72 h) = 0.50 – 1.18 mg/l
The evaluation of the last interlaboratory test arranged by Centre for Assessment of Laboratories of T.G. Masaryk Water Research Institute Prague in 2008 for potassium dichromate was performed only according growth rate. Therefore we give the results for EbC50 of the last interlaboratory test, which are stated in Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008, method C.3 - Algal Inhibition Test. The result for 72h – EbC50 was:
EbC50 (72 h) = 0.20 – 0.75 mg/l
The inhibition of test organisms in the reference substance solution meets criteria determined by the interlaboratory tests.

Table 1: Preliminary test - results

Loading rate	Mean cell density in 1 ml				Growth rate		Cell growth
mg/l	0 hours	24 hours	48 hours	72 hours	Mean growth (µ)	Reduction (%)	Mean area (A)	Inhibition (%)
100	10000	14583	179167	466667	1.28	16.8	402083	42.2
60	10000	20833	150000	531250	1.32	14.0	411458	40.9
40	10000	20833	143750	637500	1.38	10.1	458333	34.1
20	10000	18750	179167	564583	1.34	12.7	455208	34.6
10	10000	27083	135417	637500	1.38	10.1	456250	34.4
Control	10000	29167	183333	1016667	1.54	-	695833	-

Table 2: Full test - results

Loading rate	Mean cell density in 1 ml				Growth rate		Cell growth
mg/l	0 hours	24 hours	48 hours	72 hours	Mean growth (µ)	Reduction (%)	Mean area (A)	Inhibition (%)
100	10000	56250	289583	618750	1.38	14.6	630208	48.2
83	10000	54167	308333	677083	1.41	12.7	676042	44.5
69	10000	66667	327083	818750	1.47	8.8	778125	36.1
58	10000	54167	418750	856250	1.48	7.9	876042	28.1
48	10000	72917	368750	966667	1.52	5.4	900000	26.1
40	10000	68750	377083	1018750	1.54	4.3	930208	23.6
33	10000	54167	504167	1293750	1.62	-0.7	1180208	3.1
Control	10000	64583	552083	1252083	1.61	-	1217708	-

Validity criteria fulfilled:

yes

Remarks:

Exception is point 3 (the test substance concentration in test solutions should not drop under 80 % of input one during of the test). Due to low solubility the Water Accommodated Fraction method was used.

Conclusions:

These results was obtained:
EbL50 (72 h) > 100 mg/l (loading rate)
ErL50 (72 h) > 100 mg/l (loading rate)
NOELRb (72 h) = 58 mg/l (loading rate)
NOELRr (72 h) = 69 mg/l (loading rate)
The criteria for classification are given below according to article 5.2.1. of Annex IV to Commission Directive 93/21/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances.
According to the classification criteria given in quoted Directive the test substance, Slimes and Sludges, blast furnace and steelmaking, is not assigned to any of quoted toxicity categories.

Executive summary:

The test substance, Slimes and Sludges, blast furnace and steelmaking, was tested for growth inhibition on algae Desmodesmus subspicatus. The test was performed according to method C.3 - Algal Inhibition Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

Since the test substance is multi-component poorly soluble substance the water-accommodated fractions (WAFs) were prepared by adding measured amounts of the test substance to the test medium and then were stirred for a period of 72 hours. After period of stirring the WAFs were filtered through 0.45 µm filter and these solutions were used for testing. The WAFs were prepared for preliminary and full test.

The preliminary test was performed in range of test substance loading rates 10 – 100 mg/l. The inhibition of cell growth was 42.2 % at the highest test substance concentration 100 mg/l in the preliminary test. Therefore the full test was performed in appropriate loading rate range. The full test was performed in range of test substance loading rate 33 – 100 mg/l.

The study was performed without the analytical determination of the test substance concentrations. Only the concentrations of metals in the highest loading rate 100mg/l were analysed in the full test. For all evaluations and results the loading rates of the test substance were used.

Test results:

E_bL₅₀ (72 h) > 100 mg/l          (loading rate)

E_rL₅₀ (72 h) > 100 mg/l           (loading rate)

NOELR_b (72 h) = 58 mg/l      (loading rate)

NOELR_r (72 h) = 69 mg/l      (loading rate)

Classification of the test substance on the basis of hazard to aquatic environment was performed according to the Directive 93/21/EEC, Annex IV (article 5.2.1.). According to the classification criteria given in quoted Directive the test substance, Slimes and Sludges, blast furnace and steelmaking, is not assigned to any of quoted toxicity categories.

Description of key information

The test was performed according to EU Method C.3 (Algal Inhibition Test).
The study was performed without the analytical determination of the test substance concentrations. Because the test substance is multi-component poorly soluble substance the water-accommodated fractions (WAFs) were prepared for preliminary and full test. 

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

58 mg/L

Additional information

As the test substance was tested by using the water-accommodated fractions (WAFs) and nominal concentrations (loading rates) were used, EL (resp. NOELR) parameters were used instead of EC (resp. NOEC) values.

EL₅₀(72 h) > 100 mg/l.

NOELR (72 h) = 58 mg/l (biomass), NOELR (72 h) = 69 (growth rate).

For "no observed effect" requirements was used the worse value.