Germanium dioxide

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:

June-July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 1338
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store at ambient temperature; keep in original container; keep container tightly closed; keep away from food and drink; store in a dry, well-ventilated area.
- Stability under storage conditions: stable under recommended storage and handling conditions
- Stability under test conditions: stable under recommended storage and handling conditions
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: stable under recommended storage and handling conditions
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: For preparations of test item concentrations, an appropriate amount of approximately 4.61 mg test item was weighed and transferred to 1 L dilution water and afterwards stirred for 2 hours at room temperature. This solution served as highest test concentration and was serially diluted with test medium to prepare the lower test concentrations. Since undissolved test item was visible after stirring, the test solution was filtered using a 0.45 µm filter before dilution.
OTHER SPECIFICS
- other information:
Conductivity[µS/cm] 256
Alcalinity[mmol/l] 1.6
Tot. hardness[mmol/l] 1.1
Ca-hardness[mmol/l] 1.0
Mg-hardness[mmol/l] 0.1
NPOC[mg/L] 0.958
NO3[mg/L] 7.0
NO2 [mg/L] <0.005
NH4 [mg/L] <0.01
PO4 [mg/L] <0.10
Cl [mg/L] 0.02
Cd [µg/L] 0.004 - 0.005
Cr [µg/L] 0.078 - 0.082
Cu [µg/L] 1.12 - 1.17
Fe [µg/L] 0.129 - 0.217
Mn [µg/L] 0.307 - 0.328
Ni [µg/L] 0.166 - 0.172
Pb [µg/L] 0.012 - 0.013
Zn [µg/L] <0.292 (LOD)

Analytical monitoring:

yes

Details on sampling:

- Sample storage conditions before analysis: Store at ambient temperature; keep in original container; keep container tightly closed; keep away from food and drink; store in a dry, well-ventilated area.- Concentrations: Background concentrations of Ge in the controls were between 0.157 and 0.857 µg Ge/L. The five test concentrations showed mean measured concentrations of freshly prepared test solutions between 101 – 112 % of nominal concentrations. During the time interval until renewal of the test solution, test item concentrations remained stable at 94.0 – 108 % of nominal.
- Sampling method: chemical analysis of germanium by ICP-MS
- Sample storage conditions before analysis: Fresh samples, one test sample and one retain sample, of 20 mL volume were taken from all five test solutions and controls at the beginning of the test and at each media renewal prior to addition of the daphnids and feeding algae (2 x 6 samples). Aged samples were taken at each media renewal and at the end of the test from representative replicates per test concentration and control (2 x 6 samples). In order to remove feeding algae, aged samples were filtered using a 0.45 µm PES filter and afterwards, samples were immediately stabilised by adding 2.5 mL HNO3 to 20 mL test media.
Samples which were not measured on the day of sampling, were immediately stored at 4 ± 3 °C until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For preparations of test item concentrations, an appropriate amount of approximately 4.61 mg test item was weighed and transferred to 1 L dilution water and afterwards stirred for 2 hours at room temperature. This solution served as highest test concentration and was serially diluted with test medium to prepare the lower test concentrations.
- Controls: dilution water.
Purified, tap water is used as holding- and dilution water. The purification includes filtration with activated charcoal and aeration. To avoid copper contamination, plastic water pipes are used for the testing facilities.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Yes. Since undissolved test item was visible after stirring, the test solution was filtered using a 0.45 µm filter before dilution.
- Other relevant information: The test solutions were freshly prepared immediately before water renewal. For serial dilution, 357 mL test solution of the next higher concentration was given to 643 mL dilution water to prepare the lower test concentration, respectively.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: cladocerans
- Age at study initiation (mean and range, SD): 4 – 24 hours old
- Source: German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene
- Age of parental stock (mean and range, SD): 3 weeks old
- Feeding during test : suspensions of unicellular alga Desmodesmus subspicatus
- Food type: alga
- Amount: 0.2 mg C/Daphnia * Day
- Frequency: daily

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: Adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving. Batches of 30 to 50 animals were held at room temperature in ca. 1.8 L dilution water for one week. During this week, the daphnids were fed daily with an algal suspension (Desmodesmus subspicatus) and ArtemioFluid (JBL GmbH & Co. KG). Algae growing in the log-phase were centrifuged and the pellet was re-suspended in a few mL of medium. 5 mL of this suspension was given to about 1.8 L Daphnia medium. The water was changed three times per week. Newborn D. magna were separated by sieving, the first generation was discarded. Individuals applied in the test were transferred with a bore Pasteur pipette a few hours after sieving to ensure applying only healthy specimens.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

250 – 350 mg CaCO3/L

Test temperature:

19.0 – 19.7 °C

pH:

7.97 – 9.45

Dissolved oxygen:

7.60 mg/L and 13.4 mg/L

Conductivity:

256 µS/cm

Nominal and measured concentrations:

Nominal concentrations of 0.075, 0.210, 0.588, 1.65 and 4.61 mg GeO2/L
The time weighted mean concentrations of fresh and aged test solutions throughout the test were calculated to be 0.0531, 0.149, 0.429, 1.22 and 3.29 mg Ge/L (102 – 106 % of nominal), equivalent to 0.0766, 0.215, 0.619, 1.75 and 4.74 mg GeO2/L calculated based on the Ge content of 69.4% Ge in the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 50mL, /, 50mL
- Aeration: No. The beakers were covered with glass panes to prevent from evaporation as much as possible, but also permit gaseous exchange between the medium and the atmosphere and access of light.
- Renewal rate of test solution (frequency/flow rate): Test media were replaced three times per week.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate: 1 daphnid/vial

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified, tap water is used as holding- and dilution water. The purification includes filtration with activated charcoal and aeration. To avoid copper contamination, plastic water pipes are used for the testing facilities.
The following water chemistry data are recorded regularly in the testing facility and are reported: pH, conductivity, dissolved oxygen content, content of nitrate, nitrite, ammonium, phosphate, calcium, magnesium, total hardness, alkalinity, DOC content (or NPOC, as appropriate), content of metals (cadmium, chrome, copper, iron, lead, manganese, nickel and zinc).

- Non purgeable carbon: 0.958 mg/L
- Metals: Cd 0.004 - 0.005 µg/L ; Cr 0.078 - 0.082 µg/L ; Cu 1.12-1.17 µg/L ; Fe 0.129 - 0.217 µg/L ; Mn 0.307 - 0.328 µg/L ; Ni 0.166 - 0.172 µg/L ; Pb 0.012 - 0.013 µg/L ; Zn <0.292 µg/L
- Chlorine: 0.02 mg/L
- Alkalinity: 1.6 mmol/L
- Ca/mg ratio: Ca-hardness 1.0 mmol/L / Mg-hardness 0.1mmol/L
- Conductivity: 256 µS/cm

OTHER TEST CONDITIONS
- Adjustment of pH: 7.97 – 9.45
- Photoperiod: 16 hours light / 8 hours dark
- Light intensity: 771 – 973 lx (10.3 – 13.0 µE/(m² *s))

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.8
- Range finding study : Yes. In a non-GLP range-finding test, the effect of the test item on the reproduction of Daphnia magna was assessed at three nominal concentrations of 0.1, 1.0 and 10.0 mg/L. Daphnids were exposed to the test item for 21 days under semi-static test conditions with three media renewals per week. The test fulfilled the validity criteria according to OECD 211.
- Test concentrations: Nomical concentrations of (Control), 0.075, 0.210, 0.588, 1.65 and 4.61 mg GeO2/L for the present test. Nominal concentrations of 0.1, 1.0 and 10.0 mg/L for the non GLP Range Finding test.
- Results used to determine the conditions for the definitive study: In the non-GLP range finding test: At the end of the test, daphnids in the control showed 142 juveniles per parent and no parent died. The test fulfilled the validity criteria according to OECD 211.
All daphnids in the highest test concentration of 10.0 mg/L died within the 21-day test. No further mortality occurred in the lower test concentration. In addition, a significant reduction of reproduction compared to the control (28.8%) was determined for the concentration of 1.0 mg/L.

Reference substance (positive control):

yes

Remarks:

In order to confirm the sensitivity of the test species Daphnia magna, acute immobilization tests over 24 h with the reference substance (RS) K2Cr2O7 are performed in regular intervals, as proposed by OECD 202

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.619 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

GeO2

Basis for effect:

reproduction

Remarks:

of survived parent daphnids

Remarks on result:

other: 0.619 mg GeO2/L (0.429 mg Ge/L)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.619 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Remarks:

of introduced parent daphnids

Remarks on result:

other: 0.619 mg GeO2/L (0.429 mg Ge/L)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.619 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

immobilisation

Remarks:

of parent daphnids

Remarks on result:

other: 0.619 mg GeO2/L (0.429 mg Ge/L)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.619 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: length

Remarks on result:

other: 0.619 mg GeO2/L (0.429 mg Ge/L)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.619 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: development rate

Remarks on result:

other: 0.619 mg GeO2/L (0.429 mg Ge/L)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.619 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: intrinsic rate r

Remarks on result:

other: 0.619 mg GeO2/L (0.429 mg Ge/L)

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.75 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Remarks:

reproduction of survived parent daphnids

Remarks on result:

other: 1.75 mg GeO2/L (1.22 mg Ge/L)

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.75 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Remarks:

reproduction of introduced parent daphnids

Remarks on result:

other: 1.75 mg GeO2/L (1.22 mg Ge/L)

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.75 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

immobilisation

Remarks:

of parent daphnids

Remarks on result:

other: 1.75 mg GeO2/L (1.22 mg Ge/L)

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.75 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: length

Remarks on result:

other: 1.75 mg GeO2/L (1.22 mg Ge/L)

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.75 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: development rate

Remarks on result:

other: 1.75 mg GeO2/L (1.22 mg Ge/L)

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.75 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: intrinsic rate r

Remarks on result:

other: 1.75 mg GeO2/L (1.22 mg Ge/L)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 4.74 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: age of first reproduction

Remarks on result:

other: 4.74 mg GeO2/L (>=3.29 mg Ge/L)

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

4.74 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: age of first reproduction

Remarks on result:

other: 4.74 mg GeO2/L (>3.29 mg Ge/L)

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.544 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Remarks:

of survived parent

Remarks on result:

other: 0.544 mg GeO2/L (0.378 mg Ge/L)

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.585 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Remarks:

of introduced parent

Remarks on result:

other: 0.585 mg GeO2/L (0.406 mg Ge/L)

Duration:

21 d

Dose descriptor:

LC10

Effect conc.:

1.47 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

immobilisation

Remarks on result:

other: 1.47 mg GeO2/L (1.02 mg Ge/L)

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.931 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: length of parent daphnids

Remarks on result:

other: 0.931 mg GeO2/L (0.646 mg Ge/L)

Details on results:

- Other biological observations: In the test concentration of 4.74 mg GeO2/L, all exposed adult daphnids showed discoloration and were much smaller than daphnids in the control. In addition, they seemed to be less active than daphnids in the control. A significant difference between the length of daphnids in the control and the test concentrations of 1.75 mg GeO2/L was observed.
- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Some undissolved test item was visible after stirring, so the test solution was filtered using a 0.45 µm filter before dilution.

Results with reference substance (positive control):

- Results with reference substance valid? Yes for reference substance (RS) K2Cr2O7
- Relevant effect levels: EC50 = 0.6 – 2.1 mg/L
- Other: In order to confirm the sensitivity of the test species Daphnia magna, acute immobilization tests over 24 h with the reference substance (RS) K2Cr2O7 are performed in regular intervals, as proposed by OECD 202 [7]. The results of the latest reference study (March 2020) are in agreement with historical 24 h EC50-values obtained in this institute, and the stated range given in the OECD guideline (EC50 = 0.6 – 2.1 mg/L).

Immobilization after 24 h:
Control: 5 %
0.40 mg RS/L: 30 %
0.60 mg RS/L: 5 %
0.90 mg RS/L: 5 %
1.35 mg RS/L: 50 %
2.00 mg RS/L: 65 %

24 h EC50 value:
Immobilization: 1.868 mg/L (95% CL : n.d.)

Reported statistics and error estimates:

For each endpoint, the NOEC, LOEC, and the EC50 and EC10 is determined. A LOEC was calculated by using ANOVA followed by Welsh-t test, Dunnett’s or Williams’ test or an appropriate non-parametric test (Step-down Cochran-Armitage Test). Since the test results showed a concentration-response relationship, the data was analyzed by regression to determine the EC50 including the 95% confidence interval as well as the EC10 using Probit-analysis assuming log-normal distribution of the values.
The computer software ToxRat® was used for statistical evaluations.
The evaluation of the concentration-effect-relationships and the calculations of effect concentrations were based on the time weighted mean (TWM) concentrations, since the measured concentrations varied by more than 20% from the nominal concentrations throughout the test.

All statistical calculations were based on time weighted mean (TWM) concentrations of the test item. The coefficient of variation of cumulative offspring per survivor was 14.6 %. The Cochran-Armitage trend-test revealed a significant trend in mortality. According to OECD 211 it is required to report the NOEC|EC for the cumulative offspring per introduced parent provided these values are lower than in the cumulative offspring per survivor. In the present test, the EC10 is lower for the offspring per survived parent. Therefore, the relevant endpoint is the NOEC|EC for the cumulative offspring per survived parent daphnid. Nevertheless, both endpoints (offspring per introduced and survived parent) are reported.
To be valid, the maximum control mortality in females must not exceed 20% and the mean offspring number at day 21 must be at least 60.0. In present test mortality was 0% and mean offspring number was 114.3. With respect to all these criteria the test is valid.

Validity criteria fulfilled:

yes

Conclusions:

Tests done according to standard protocol.

Following a 21-day exposure period, a NOEC of 0.429 mg Ge/L and a LOEC of 1.22 mg Ge/L were determined, for all considered endpoints, established based on TMW concentrations.
The age of first reproduction showed no effect, resulting in a NOEC of ≥3.29 mg Ge/L and a LOEC of > 3.29 mg Ge/L.
 
An EC10 of 0.378 mg Ge/L and 0.406 mg Ge/L was calculated for reproduction of survived and introduced parent, respectively. A concentration-response was observed for immobilization, resulting in a LC10 of 1.02 mg Ge/L. For the length of parent daphnids at test end an EC10 of 0.646 mg Ge/L.

Executive summary:

Tests done according to standard protocol.

Following a 21-day exposure period, a NOEC of 0.429 mg Ge/L and a LOEC of 1.22 mg Ge/L were determined, for all considered endpoints, established based on TMW concentrations.

The age of first reproduction showed no effect, resulting in a NOEC of ≥3.29 mg Ge/L and a LOEC of > 3.29 mg Ge/L.

 

An EC10 of 0.378 mg Ge/L and 0.406 mg Ge/L was calculated for reproduction of survived and introduced parent, respectively. A concentration-response was observed for immobilization, resulting in a LC10 of 1.02 mg Ge/L. For the length of parent daphnids at test end an EC10 of 0.646 mg Ge/L.

Description of key information

The influence of the test item germanium dioxide (GeO2) on the reproduction of aquatic invertebrates, represented by Daphnia magna, was investigated. A 21-day semi-static exposure to five concentrations of the test item according to the OECD guideline 211 was performed.
During the 21-day exposure period, the test item had significant effects on the investigated endpoints of reproduction, immobilisation, length, development rate and intrinsic rate r of the test organisms Daphnia magna.
Calculated EC10 values were lower for the cumulative offspring per survivor than per introduced daphnid and, thus, the relevant endpoint is the reproduction based on the cumulative offspring per survivor.
The EC10 for reproduction per survived parent was at 0.544 mg GeO2/L (0.378 mg Ge/L). The LC10 for immobility was at 1.470 mg GeO2/L (1.02 mg Ge/L). Based on the evaluation performed on TWM concentrations of GeO2 calculated based on the measured Ge concentrations in aqueous samples, for reproduction per survived and introduced parent daphnid, immobility, development rate and intrinsic rate r, a NOEC of 0.619 mg test item/L and a LOEC of 1.75 mg test item /L were determined. Considering the content of Ge in GeO2 of 69.4 %, the NOEC and LOEC are at 0.429 mg Ge/L and 1.22 mg Ge/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

0.544 mg/L