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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September-October 2012
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Sulfite liquors and Cooking liquors, green
EC Number:
268-612-2
EC Name:
Sulfite liquors and Cooking liquors, green
Cas Number:
68131-30-6
Molecular formula:
HNa3OS
IUPAC Name:
Aqueous alkaline solution of sodium salts produced by dissolving melt from incineration of spent liquor from sodium based pulping processes.
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Name of test material (as cited in study report): Green liquor (sulphite process)
- Substance type: UVCB
- Physical state: greenish liquid
- Composition of test material, percentage of components: 19.6 % dry matter, water content 80.4 %
- Name of test material (as cited in study report): Sulfite liquors and Cooking liquors, green
- Substance type:unknown or variable composition (UVCB)
- Physical state: liquid (dissolved solids in water, contains always water 75 % or more )
- Vapour pressure at 100°C: 2425 Pa
- Relative density (20°): 1,15
- Water miscibility: Totally soluble in water
- Impurities (identity and concentrations): not relevant
- Composition of test material, percentage of components:

Analyte Method Unit Result
Sodium Na SCAN-N 38:10 modif g/l 93
Potassium K SCAN-N 38:10 modif g/l 1,3
Sulfur S SCAN-N 38:10 modif g/l 57
Thiosulfate S2O3= KCL 70:83 modif. g/l 5,7
Sulphate SO42- SCAN-N 6:85 g/l 16,1
Effective alkali NaOH SCAN-N 30:85 g/l 59
Active alkali NaOH SCAN-N 30:85 g/l 120
Total alkali NaOH SCAN-N 30:85 g/l 150
Sodium sulfide Na2S calculated g/l 122
Sodium carbonate Na2CO3 calculated g/l 42,9
Sodium hydroxide NaOH calculated g/l n.d
Sodium thiosulfate Na2S2O3 calculated g/l 7,9
Potassium hydroxide KOH calculated g/l n.d
Potassium carbonate K2CO3 calculated g/l 0,5
Potassium sulfide K2S calculated g/l 1,4
Potassium thiosulfate K2S2O3 calculated g/l 0,08


- Analysis date and laboratorium: 28.8-12.9.2012, Mia Tehomaa, Labtium Ltd, Tekniikantie 2, 02150 Espoo, Finland (lab reporrt 113630)
- Lot/batch No.: 2012-v32 GL1 after sedimentation and filtration
- Stability under test conditions: Stable at least until August 2015
- Storage condition of test material: Sealed PE Plastic containers, 2-8 °C, dark

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): M7 media (composition, see OECD TG no. 211 (table 2))
M7-media was prepared according to OECD TG no. 211 (table 2). Due to the presence of metals in the test item, Fe-EDTA was excluded from the M7-media because the EDTA could potentially bind to the metals, and thereby disguise a possible toxicity. The range finding test made by Toxicon included a test using two types of M7-media, with and without Fe-EDTA which confirmed that the exclusion of Fe-EDTA would not affect the potential toxicity results. The M7-media had a pH of 7,8±0,2 and a theoretical hardness of 250±25 mg/l (expressed as CaCO3).
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No

A fresh stock solution with a concentration of 100 mg/l was made at every water renewal. M7-media was used as dilution solution. The stock solution was diluted using aerated M7-media and feed (green algae) to obtain the test concentrations 1,5 ; 3,0 ; 6,0 ; 12 ; 24 and 48 mg/l (wet weight) .The concentrations above are equal to a dry weight of 0,29 ; 0,59 ; 1,2 ; 2,4 ; 4,7 and 9,4 mg/l. The chosen test concentrations are lower than what the range finding test indicated due to an unexpected high mortality in the first reproduction test.

pH were not adjusted in any of the test concentrations, and the M7-media was aerated prior to water renewals.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Clone-A (identified by genotyping)
- Source: Norwegian Institute of Water Research (NIVA), Norway
A qualification test on the Daphnia magna culture was made on the 28th of June 2012 to assure the quality of the test animals using K2Cr2O7. EC50 was 1,1 mg/l, which confirmed that the culture was suitable as a test organism according to OECD TG no. 202. On the 12th of July 2012 a qualification test was performed using a dilution solution without Fe-EDTA, resulting in an EC50 of 1,0 mg/l.
- Feeding during main test, yes
- Food type: The adult animals were fed with the green algae Pseudokirchneriella subcapitata when the media was renewed.The algal feed was concentrated to make sure the added volume of feed was as low as possible.
- Amount: The amount of algae added corresponded to 0,2 mg of organic carbon per animal and day.
- Frequency: Three times a week when the media was renewed (a semi static test, the media was renewed three times a week)

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d

Test conditions

Hardness:
Theoretical hardness 250±25 mg/l (as CaCO3). Hardness was not measured.
Test temperature:
19-21° C
pH:
Test M7 media pH adjusted to 7,8±0,2
Test solutions measured pH: 7.5-9.4
Control measured pH: 7.8-9.5
Dissolved oxygen:
Control:
Test solutions: 100-139 %
Nominal and measured concentrations:
1,5 ; 3,0 ; 6,0 ; 12 ; 24 and 48 mg/l (wet weight, nominal) .The concentrations are equal to a dry weight of 0,29 ; 0,59 ; 1,2 ; 2,4 ; 4,7 and 9,4 mg/l.The dry solids content of the tested sample is 19.6% dry solids/80.4% water by weight (at 20 deg. C))
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 ml glass test vessels
- Type: open
- Material, size, headspace, fill volume:
- Aeration: No

- Renewal rate of test solution (frequency/flow rate): three times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10
- Biomass loading rate: The amount of algae (Pseudokirchneriella subcapitata) added corresponded to 0,2 mg of organic carbon per animal and day. The added feed volume, 3 % v/v of the test solution, was constant during the entire test.
The adult animals were fed with the green algae when the media was renewed. The algal feed was concentrated to make sure the added volume of feed was as low as possible.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M7-media (see 211 guideline)
- Total organic carbon: Not determined
- Particulate matter: No
- Metals: Not determined

- Intervals of water quality measurement: New solutions were made nine times during the reproduction test.

RANGE-FINDING STUDY
- Control: Two sets of M7 media control 21 day studies were carried out with no added GL. First (1.) with Fe-EDTA and second without Fe-EDTA. There was no statistical difference on the statistical output between the reproduction results (Dunnet's p < 0.05), mean of living offspring produced per parent animal.

(1) 148 (±26) offspring with Fe-EDTA
(2) 158 (±23) offspring without Fe-EDTA

- Range finding short term (48 hr) toxicity test concentrations: Loading rates, two sets of range finding studies were carried out, first without Fe-EDTA and second with Fe-EDTA.

The M7 solution without Fe-EDTA was 4-6 times more toxic than with Fe-EDTA. The 48 hr NOEC was 6.3 mg/L (substance dry weight) without and 39 mg/l (dry weight) with Fe-EDTA. Test concentration in the range-finding study were 2.5, 6.3, 16, 39 and 98 mg/l (dry weight), 12.8, 32, 80, 200 and 500 mg/l (wet weight)

- Results used to determine the conditions for the definitive study: M7 solution without Fe-EDTA was selected for the final reproduction study.
- Study protocoll:
Reference substance (positive control):
yes
Remarks:
An acute qualification test on the Daphnia magna culture was made to assure the quality of the test animals using K2Cr2O7. EC50 was 1,1 mg/l, which confirmed that the culture was suitable as a test organisms according to OECD 202.

Results and discussion

Effect concentrationsopen allclose all
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
2.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
dry weight
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
2.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
dry weight
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
4.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
dry weight
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
4.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
dry weight
Basis for effect:
mortality
Remarks:
of parent animals
Details on results:
- Mortality of parent animals: In the highest test concentration 9.4 mg/l (dry weight/l) the parent animals died early on. On the last day of the test (day 21) six out of the ten parent animals had died in the second highest test concentration (4,7 mg dry weight/l) and none had died in the rest of the test concentrations.
- No. of offspring produced per day per female:
Controll: 153±23 offspring
136±16 at 0.29 mg/l (dw)
134±16 at 0.59 mg/l (dw)
146±15 at 1.2 mg/l (dw)
137±15 at 2.4 mg/l (dw)
138±41 at 4.7 mg/l (dw)
0 at 9.4 mg/l (dw)

- Time to first brood release or time to hatch:Time to first brood was between 7 and 8 days in all of the test concentrations, except in the highest test concentration 9.4 mg/l where the parent animals died early on.

Reported statistics and error estimates:
The Lowest Observed Effect Concentration (LOEC), the lowest concentration with a statistical difference to the control (p <0,05), was determined using a one-way ANOVA (Dunnett´s) on the parameter number of living offspring per parent animal. The No Observed Effect Concentration (NOEC) is according to OECD TG no. 211 the concentration below LOEC.

Any other information on results incl. tables

The criteria for validity of the test according to OECD TG no. 211 were fulfilled:

•The mean number of live offspring produced per parent animal in the control surviving at the end of the test was ≥60 (table2).

•The mortality of parent animals in the control did not exceed 20% at the end of the test (table 2).

•The coefficient variation around the mean number of living offspring produced per parent animal in the control was ≤ 25 % (in this test it was calculated to be 15 %).

Time to first brood was between 7 and 8 days in all of the test concentrations, except in the highest test concentration where the parent animals died early on. On the last day of the test (day 21) six out of the ten parent animals had died in the second highest test concentration (4,7 mg dry weight/l) and none had died in the rest of the test concentrations. All of the parent animals had died in the highest test concentration (9,4 mg dry weight/l). There was no mortality amongst the offspring.

Table 1. NOEC- and LOEC-values (mg/l) for the reproduction (living offspring per parent animal) after 21 days of exposure to the test item Green Liquor (GL).

 

21 days NOEC (mg/l)

21 days LOEC (mg/l)

Reproduction, dry weight

2,4

4,7

Reproduction, wet weight

12

24

Mortality (parent animal), dry weight

2,4

4,7

Mortality (parent animal), wet weight

12

24

Table 2. Living offspring produced per parent animal (mean± standard deviation) and the number of dead parent animalsafter 21 days of exposure to the test item Green Liquor (GL).

Test conc.

dry weight

(mg/1)

Test conc.

wet weight

(mg/1)

Mean ofliving

offspring / parent animal

Number of dead parent animals / total

control

control

153±23

0/10

0,29

1,5

136±16

0/10

0,59

3,0

134±16

0/10

1,2

6,0

146±15

0/10

2,4

12

137±15

0/10

4,7

24

138±41

6/10

9,4

48

-

10/10

Applicant's summary and conclusion