Vinasses, residue of fermentation

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 June 2010 - 13 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10706: “Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea)”, 2000-03-30.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 40 mL samples for TOC-analysis were taken from the test concentration and the control at the beginning and at the end of two intervals of 72 hours (nominal days 0 and 3, 17 and 20) and two intervals of 48 hours (nominal days 6 and 8, 13 and 15).
- Sample storage conditions before analysis: Samples not analysed on the day of sampling were stored in a refrigerator analysis.

Additionally, reserve samples of 40 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The batch of Vinasses tested was a dark brown suspension that was completely soluble in test medium at the concentration tested.
Three times a week a loading rate of 100 mg/L was magnetically stirred for 15 minutes to accelerate the dissolving of the test substance in the test medium. The final test solution was clear and yellow. Lower test concentrations were prepared by subsequent dilutions of the 100 mg/L concentration in test medium.
- Controls: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea, Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Source: In-house laboratory culture with a known history.
- Age at study initiation: < 24 hours
- Method of breeding: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 10 litres of medium in an all-glass culture vessel.
- Food type: Chlorella pyrenoidosa suspension / Algae suspensions were made by inoculating growth medium with Chlorella pyrenoidosa from a pure culture. The suspensions were continuously aerated and exposed to light (6000-10000 lux; 60-120 µE.m-2.s-1) in a climate room at a temperature of 23 ± 2 °C for 2-3 weeks. After this period, the TOC concentration of the batch algal suspension was measured before use as feed in the reproduction study. The exact volume to be added per test vessel was then calculated.
- Amount: 0.25 mL; This daily ration corresponded to 0.2 mg C/Daphnia/day, which is the recommended value for daily feeding per daphnid in the reproduction test according to the OECD Guideline 211.
- Frequency: daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

between 201 and 254 mg calcium carbonate per litre

Test temperature:

The temperatures in the test media varied between 19.8 and 21.0 °C. The temperature, continuously measured in a temperature control vessel varied between 20.1 and 21.7 °C during the test.

pH:

The pH remained within the range of 7.4 to 8.3 throughout the test.

Dissolved oxygen:

The oxygen concentration in all test solutions remained within the range of 6.3 to 9.6 mg/L during the exposure period.

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations: control and 10, 18, 32, 56 and 100 mg/L
Measured concentrations: Based on the TOC content of Vinasses (i.e. 25.96%) the concentration of Vinasses in the aquatic samples was calculated and corrected for the control. The initial concentrations were below nominal, i.e. between 35 and 46%. These concentrations remained stable during the respective refreshment periods, i.e. between 82 and 123% of initial. Based on these results the average exposure concentrations were calculated to be 3.8, 7.4, 13, 23 and 42 mg/L.

Details on test conditions:

- Type (delete if not applicable): volumetric flasks
- Material, size, headspace, Volume: 60 mL (6 x Ø 3.5 cm), all-glass covered with a Perspex plate
- Renewal rate of test solution (frequency/flow rate): three times a week
- Aeration: no
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20
- Extra replicate vessels without daphnids were included for sampling purposes.
- Feeding during test: Daily, 0.25 mL of Chlorella pyrenoidosa suspension containing 1.3 x10E8 cells was added as feed for the daphnids. This daily ration corresponded to 0.2 mg C/Daphnia/day, which is the recommended value for daily feeding per daphnid in the reproduction test according to the OECD Guideline 211

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
Macro salts:
CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCl: 4.2 mg/L

Medium M7: trace elements, macro nutrients and vitamins are added to freshly prepared ISO medium to reach the following concentrations:
Trace elements:
B: 0.125 mg/L
Fe:0.05 mg/L
Mn: 0.025 mg/L
Li, Rb and Sr: 0.0125 mg/L
Mo: 0.0063 mg/L
Br: 0.0025 mg/L
Cu: 0.0016 mg/L
Zn: 0.0063 mg/L
Co and I: 0.0025 mg/L
Se: 0.0010 mg/L
V: 0.0003 mg/L
Na2EDTA.2H2O: 2.5 mg/L

Macro nutrients:
Na2SiO3. 9H2O: 10.0 mg/L
NaNO3: 0.27 mg/L
KH2PO4: 0.14 mg/L
K2HPO4:0.18 mg/L

Vitamins:
Thiamine: 75.0 µg/L
B12: 1.0 µg/L
Biotin: 0.75 µg/L

The hardness: 180 mg/L expressed as CaCO3 and the pH: 7.7 ± 0.3 after aeration.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light/8 h dark
intensity at the start: 663-711 lux (9.2-9.8 µE.m-2.s-1)
intensity at the end: 629-736 lux (8.1-9.2 µE.m-2.s-1)

EFFECT PARAMETERS MEASURED
Parental daphnids
Condition: Every workday and upon renewal on non-workdays, the number of living, immobile and dead parental daphnids was recorded. Dead daphnids were removed when observed.
Presence of eggs in the brood pouch: Every workday and upon renewal on non-workdays.
Body length: At the end of the test.

F1 generation
Appearance first brood: When observed.
Newborn daphnids: Every workday and upon renewal on non-workdays, the number of newborn young was counted and the condition of the young recorded. Thereafter the young were removed.
Presence of unhatched eggs: When observed.
Incidence of immobility: When observed.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Test concentrations: control and 10, 18, 32, 56 and 100 mg/L

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

: Parental mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

: Parental mortility

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

: Parental mortality

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

: Parental body length

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

: Parental body length

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Overall LOEC

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Overall NOEC

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

32 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Remarks:

Total Organic Carbon (TOC) converted to test material

Basis for effect:

mortality

Remarks:

Parental mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Remarks:

Total Organic Carbon (TOC) converted to test material

Basis for effect:

mortality

Remarks:

Parental mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

23 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Remarks:

Total Organic Carbon converted to test material

Basis for effect:

mortality

Remarks:

Parental mortality

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Remarks:

Toatl Organic Carbon (TOC) converted to test material

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Remarks:

Toatal Organic Carbon (TOC) converted to test material

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Remarks:

Total Organic Carbon (TOC) converted to testmaterial

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Remarks:

Total Organic Carbon (TOC) converted tot test material

Basis for effect:

growth

Remarks:

Parental body length

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Remarks:

Total Organic Carbon (TOC) converted tot test material

Basis for effect:

growth

Remarks:

Parental body length

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

42 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Remarks:

Total organic Carbon (TOC) convereted tot test material

Basis for effect:

other: Overall LOEC

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

23 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Remarks:

Total Organic Carbon (TOC) converetd to test material

Basis for effect:

other: Overall NOEC

Details on results:

Mortality of parental animals:
Control: 3 out of 20 (15%)
10 mg/L: 2 out of 10 (20%)
18 mg/L: 2 out of 10 (20%)
32 mg/L: 1 out of 10 (10%)
56 mg/L: 4 out of 10 (40%)
100 mg/L: 6 out of 10 (60%)

Mean number of offspring:
Control: 132.6 ± 16.3 per 21 days
10 mg/L: 164.8 ± 11.0 per 21 days
18 mg/L: 182.6 ± 21.9 per 21 days
32 mg/L: 202.4 ± 17.9 per 21 days
56 mg/L: 200.0 ± 29.8 per 21 days
100 mg/L: 141 ± 54.1 per 21 days

Body lenght of parent animals:
Control: 4.26 (n=17) ± 0.12 mm
10 mg/L: 4.35 (n=8) ± 0.10 mm
18 mg/L: 4.33 (n=8) ± 0.14 mm
32 mg/L: 4.37 (n=9) ± 0.16 mm
56 mg/L: 4.29 (n=6) ± 0.12 mm
100 mg/L: 4.02 (n=4) ± 0.25 mm

Type and number of morphological abnormalities: Not observed.
Type and number of behavioral abnormalities: Not observed
Time to first brood release: the first brood generally appeared on day 8
The presence of eggs in the brood pouch: the first time on day 6. At nominal 100 mg/L the first presence of eggs in the brood pouch was similar or slightly later (day 6 or day 7). There were no recordings of immobile young or appearance of unhatched (aborted) eggs at any of the test concentrations throughout the 21-day test period.

Reported statistics and error estimates:

REPRODUCTION
Statistical analysis for reproduction was not performed as no adverse effects were noted in the treatment groups in comparison to the control
Mortality was statistically significant at nominal 100 mg/L (Fisher’s exact test, α=0.05)

PARENTAL DAPHNIDS
Parental survival on day 21 was statistically tested using Fisher’s exact test (TOXSTAT Release 3.5, 1996, D.D. Gulley, A.M. Boelter, H.L. Bergman).

The overall threshold level of effect and the overall NOEC were determined on basis of these results and statistics. The LC50 for mortality of parental daphnids was determined using the maximum likelihood estimation method with the probits of the percentages of dead daphnids as function of the logarithms of the corresponding concentrations (Finney, D.J., 1971: Probit analysis, Cambridge University Press, Cambridge, U.K., 3rd edition).

21-day LC50 parental daphnids = 77.4 mg/L (based on mean concentrations)
95 % fiducial limits: 51.6 - 417.2 mg/L
index of regression significance: g=0.73
chi-squared=0.27, with 1 degrees of freedom
regression line: log10(conc.)=1.78+(probit-4.69)/2.96

21-day LC50 parental daphnids = 32.2 mg/L (based on nominal concentrations)
95 % fiducial limits: 21.2 - 184.5 mg/L
index of regression significance: g=0.73
chi-squared=0.28, with 1 degrees of freedom
regression line: log10(conc.)=1.40+(probit-4.69)/2.87

Statistical analysis of the data on body lenght was not needed as the % reduction recorded was not significant (< 10%).

Vinasses did not affect reproduction of Daphnia magna at an average exposure concentration 42 mg/L (= 100 mg/L nominal) (NOEC) based on the average measured concentration after 21 days. Further effect parameters measured are presented in table 1.

Table 1: Effect parameters

Parameter	Nominal conc. Vinasses (mg/L)	Average conc. Vinasses (mg/L)
21-day LC50 (parental mortality)^1	77	32
LOEC for parental mortality^2	100	42
NOEC for parental mortality^2	56	23
21-day EC50 for reproduction	>100	>42
21-day LOEC for reproduction	>100	>42
21-day NOEC for reproduction	100	42
21-day LOEC for parental body length	>100	>42
21-day NOEC for parental body length	100	42
Overall LOEC	100	42
Overall NOEC	56	23

¹Finney probit analysis

² Fisher’s exact test (Toxstat)

Table 2: Cumulative scores of living newborn per parent after 21 days (based on nominal concentrations)

Parent (Control)	Day 21	Parent (10 mg/L)	Day 21	Parent (18 mg/L)	Day 21	Parent (32 mg/L)	Day 21	Parent (56 mg/L)	Day 21	Parent (100 mg/L)	Day 21
P1	+	P1	+	P1	+	P1	195	P1	167	P1	+
P2	116	P2	176	P2	138	P2	197	P2	203	P2	+
P3	124	P3	153	P3	186	P3	209	P3	187	P3	+
P4	148	P4	182	P4	171	P4	210	P4	+	P4	91
P5	141	P5	173	P5	191	P5	193	P5	233	P5	+
P6	159	P6	164	P6	215	P6	236	P6	237	P6	134
P7	154	P7	153	P7	181	P7	169	P7	173	P7	124
P8	+	P8	+	P8	+	P8	+	P8	+	P8	+
P9	159	P9	159	P9	190	P9	204	P9	+	P9	+
P10	+	P10	158	P10	189	P10	209	P10	+	P10	218
P11	115
P12	113
P13	127
P14	114
P15	124
P16	130
P17	116
P18	146
P19	142
P20	126
Mean	132.6		164.8		182.6		202.4		200.0		141.8
SD	16.3		11.0		21.9		17.9		29.8		54.1
n	17		8		8		9		6		4

+: Parental daphnid was found dead

Table 3: Average body lengths (mm) of parental daphnids at the end of the test

Nominal conc. Vinasses (mg/L)	Average length (mm)	SD	% Reduction
control	4.26  (n=17)	0.12	-
10	4.35  (n=8)	0.10	-2
18	4.33  (n=8)	0.14	-2
32	4.37  (n=9)	0.16	-3
56	4.29  (n=6)	0.12	-1
100	4.02  (n=4)	0.25	6

n = number of daphnids surviving the test period

SD = standard deviation

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present test Vinasses did not affect reproduction of Daphnia magna at a nominal concentration of 100 mg/L after 21 days of exposure (NOEC), corresponding to an average exposure concentration of 42 mg/L.

Description of key information

NOECreproduction (21d) > 100 mg/L (nominal), > 42 mg/L (measured)

NOECparental mortality (21d) = 56 mg/L (nominal), = 23 mg/L (measured)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

23 mg/L

Additional information

A long-term study with Daphnia magna was performed to investigate the toxicity of Vinasses towards aquatic invertebrates. The study was conducted according to the OECD Guideline 211 “Daphnia magna Reproduction Test” in a semi-static test design for 21 days. Vinasses, residue of fermentation was applied in concentrations of 10, 18, 32, 56 and 100 mg/L. Based on the TOC content of the test material (i.e. 25.96%), the concentration of Vinasses in the aquatic samples was calculated and corrected for the control, resulting in average exposure concentrations of 3.8, 7.4, 13, 23 and 42 mg/L. No chronic effects on reproduction were observed at the highest test concentration of 42 mg/L, based on measured concentrations, corresponding to 100 mg/L, nominal concentration. The lowest no observed effect concentration was obtained for parental mortality: NOEC = 23 mg/L measured (56 mg/L nominal).